Apparatus for making gas-filled vesicles of optimal size

ABSTRACT

A method and apparatus for making vesicles suitable for use as contrast agents in which a container containing an aqueous suspension phase and a separate gas phase is shaken using reciprocating motion. The reciprocating motion is produced by a shaker arm that moves the container in two, substantially perpendicular directions, with the motion in the first direction being along an arcuate path. The overall path of the motion occurs in a figure-8 eight pattern. The frequency of shaking is at least approximately 2800 RPM, the length of the shaker arm is at least approximately 6 cm, and the angle through which the shaker arm rotates in the first direction is at least approximately 3 DEG . The total length of travel around the figure-8 pattern is at least 0.7 cm.

RELATED APPLICATIONS

This application is a divisional of U.S. Ser. No. 08/482,294, filed Jun.7, 1995, now U.S. Pat. No. 5,656,211. U.S. Ser. No. 08/482,294 is acontinuation-in-part of U.S. Ser. No. 08/307,305, filed Sep. 19, 1994,and now U.S. Pat. No. 5,773,024, which in turn is a continuation-in-partof application U.S. Ser. No. 08/159,687, filed Nov. 30, 1993, now U.S.Pat. No. 5,585,112, which is in turn a continuation-in-part ofapplication U.S. Ser. No. 08/076,239, filed Jun. 11, 1993, now U.S. Pat.No. 5,469,854. U.S. Ser. No. 08/482,294 is also a continuation-in-partof U.S. Ser. No. 08/160,232, filed Nov. 30, 1993, now U.S. Pat. No.5,542,935, which in turn is a continuation-in-part of application Ser.No. 08/076,250, filed Jun. 11, 1993, now U.S. Pat. No. 5,580,575. U.S.Ser. No. 08/076,239 and U.S. Ser. No. 08/076,250 are each continuationsin part of application U.S. Ser. No. 07/717,084, now U.S. Pat. No.5,228,446, and application U.S. Ser. No. 07/716,899, now abandoned, bothof which were filed Jun. 18, 1991, which in turn arecontinuation-in-parts of application U.S. Ser. No. 07/569,828, filedAug. 20, 1990, now U.S. Pat. No. 5,088,499, which in turn is acontinuation-in-part of application U.S. Ser. No. 07/455,707, filed Dec.22, 1989, now abandoned.

FIELD OF THE INVENTION

The current invention is directed to a method and apparatus for makinggas-filled vesicles, especially gas-filled vesicles of the type usefulfor ultrasonic imaging. More specifically, the current invention isdirected to a method and apparatus for making gas-filled vesicles byshaking in which the shaking parameters are controlled to providevesicles of optimum size in a minimum amount of time.

BACKGROUND OF THE INVENTION

Ultrasound is a diagnostic imaging technique which provides a number ofadvantages over other diagnostic methodology. Unlike techniques such asnuclear medicine and x-rays, ultrasound does not expose the patient topotentially harmful exposures of ionizing electron radiation that canpotentially damage biological materials, such as DNA, RNA, and proteins.In addition, ultrasound technology is a relatively inexpensive modalitywhen compared to such techniques as computed tomography (CT) or magneticresonance imaging.

The principle of ultrasound is based upon the fact that sound waves willbe differentially reflected off of tissues depending upon the makeup anddensity of the tissue or vasculature being observed. Depending upon thetissue composition, ultrasound waves will either dissipate byabsorption, penetrate through the tissue, or reflect back. Reflection,referred to as back scatter or reflectivity, is the basis for developingan ultrasound image. A transducer, which is typically capable ofdetecting sound waves in the range of 1 MHz to 10 MHz in clinicalsettings, is used to sensitively detect the returning sound waves. Thesewaves are then integrated into an image that can be quantitated. Thequantitated waves are then converted to an image of the tissue beingobserved.

Despite technical improvements to the ultrasound modality, the imagesobtained are still subject to further refinement, particularly inregards to imaging of the vasculature and tissues that are perfused witha vascular blood supply. Hence, there is a need for the formulation ofagents that will aid in the visualization of the vasculature andvascular-related organs.

Vesicles are desirable as contrast agents for ultrasound because thereflection of sound at a liquid-gas interface, such as the surface of avesicle, is extremely efficient.

To be effective as ultrasound contrast agents, the vesicles should be aslarge and elastic as possible since both these properties (bubble sizeand elasticity) are important in maximizing the reflectivity of soundfrom the vesicles. Additionally, the vesicles should be stable topressure, i.e. retain more than 50% of the gas content after exposure topressure. It is also highly desirable that the vesicles should re-expandafter the release of pressure. Further, it is highly desirable to have ahigh vesicle concentration in order to maximize reflectivity and, hence,contrast. Therefore, vesicle concentration is an important factor indetermining the efficacy of the vesicles. In particular, it is desirableto have more than 100×10⁶ vesicles per mL and, more preferably, morethan 500×10⁶ vesicles per mL.

Size, however, remains a crucial factor in determining the suitabilityof vesicles for imagining. In the regime of vesicles that can passsafely through the capillary vasculature, the reflected signal (RayleighScatterer) can be a function of the diameter of the vesicles raised tothe sixth power so that a 4 μm diameter vesicle may possess 64 times thescattering capability of a 2 μm diameter vesicle.

Size is also important because vesicles larger than 10 μm can bedangerous. Large vesicles have a tendency to occlude micro-vesselsfollowing intravenous or intravascular injection. Hence, it is importantthat the vesicles be as large as possible to efficiently reflect soundbut small enough to pass through the capillaries.

In this regard, it is highly desirable that 99% of the vesicles besmaller than 10 μm. Further, the mean vesicle size should be at least0.5 μm, preferably over 1 μm, and more preferably close to 2 μm for mosteffective contrast. In addition, the volume weighted mean should be onthe order of 7 μm.

The elasticity of the vesicles may affect their maximum permissible sizesince the greater the elasticity of the vesicle, the greater its abilityto "squeeze" through capillaries. Unfortunately, a number of factors mayprevent the formation of highly elastic vesicles, thereby furtherreenforcing the importance of optimizing vesicle size.

While uncoated vesicles have maximal elasticity, they are generallyunstable. Consequently, efforts are often undertaken to improve thestability of the vesicles, such as by coating, that have the effect ofreducing their elasticity. In addition, the use of gas or gas-precursorsencapsulated in a proteinaceous shell, with the protein beingcross-linked with biodegradable cross-linking agents, has beensuggested, as well as the use of non-proteinaceous vesicles cross-linkedcovalently with biocompatible compounds. It may be assumed that suchcross-linkers will add a component of rigidity to the vesicles, thusreducing their elasticity.

While it is known that liposomes can be made by shaking a solution ofsurfactant in a liquid medium (see, U.S. Pat. No. 4,684,479 (D'Arrigo)),a method for making vesicles having optimal size in a minimal amount oftime has not heretofore been developed. Consequently, for all of theforegoing reasons, there is a need for a method and apparatus for makingvesicles in which the shaking parameters are controlled so as to producevesicles of optimum size in a minimum amount of time.

SUMMARY OF THE INVENTION

It is an object of the current invention to provide a method andapparatus for making vesicles in which the shaking variables arecontrolled so as to produce vesicles of optimum size in a minimum amountof time. This and other objects is accomplished in a method in which acontainer containing an aqueous suspension phase and a gas phase isshaken using reciprocating motion. The reciprocating motion is producedby a shaker arm that moves the container in two, substantiallyperpendicular directions. The motion in the first direction occurs alongan arcuate path having a radius of curvature of at least 6 cm andencompasses an angle of at least 3°. The overall path of the motionoccurs in a figure-8 eight pattern. The frequency of shaking is at least2800 RPM, the amplitude of the shaking is at least 0.3 cm and the totallength of travel of the container during each cycle is at least 0.7 cm.

The current invention also encompasses an apparatus for shaking acontainer containing an aqueous suspension phase and a gas phase usingthe method described above.

Preferably, the apparatus has a shaker arm having a length of at least 6cm that rotates through an angle of at least 3°.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a elevation of the container portion of the shaking apparatusof the current invention, in which vesicles are made by the shakingmethod of the current invention.

FIG. 2 is an isometric view of the shaking apparatus according to thecurrent invention, without the container.

FIG. 3 is a longitudinal cross-section through the shaking apparatusshown in FIG. 2, without the cover, but including the installation ofthe container shown in FIG. 1.

FIGS. 4 and 5 are elevation and plan views, respectively, of the pathtaken by the container shown in FIG. 1 when it is installed on theshaking apparatus shown in FIG. 2, with FIG. 5 being taken along theline V--V shown in FIG. 4.

FIG. 6 is an isometric view of the major internal components of theshaking apparatus shown in FIG. 2.

FIGS. 7 and 8 are longitudinal cross-sections through the shakingapparatus shown in FIG. 2 in the vicinity of the region where the shakerarm is mounted onto the motor shaft, with the position of the shaker armwhen the eccentric bushing is in the orientation shown in FIG. 8 beingshown in phantom in FIG. 7.

FIGS. 9(a) and (b) are views taken along line IX--IX shown in FIG. 7 ofthe shaker arm, except that in FIGS. 9(a) and (b) the eccentric bushinghas been rotated 90° and 270°, respectively, from its orientation shownin FIG. 7.

FIG. 10 is a cross-section taken through line X--X shown in FIG. 9(b),with the orientation of the sleeve when the eccentric bushing has beenrotated 180° shown in phantom.

FIG. 11 is an isometric view of the eccentric bushing as mounted on themotor shaft.

FIG. 12 is a view similar to FIG. 9 showing the orientation of theshaker arm when lower spring tension is employed.

FIG. 13 is a chart showing the relationship between the shakingfrequency, in RPM, on the one hand, and the shaker arm length L, in cm,and bearing offset angle θ, on the other hand, used to obtain the testresults shown in FIGS. 14-16.

FIGS. 14(a)-(c) are charts showing the percentage of the vesicles havinga size less than 10 μm, the number weighted mean size, and the particlesper mL, versus the shaker arm length L, in mm, as the shaker arm lengthand RPM are varied in accordance with FIG. 13, at a bearing offset angleθ of 6°.

FIGS. 15(a)-(c) are charts similar to FIGS. 14(a)-(c) comparing theresults obtained using a bearing offset angle θ of 9° to those shown inFIGS. 14(a)-(c).

FIGS. 16(a)-(c) are charts showing the percentage of the vesicles havinga size less than 10 μm, the number weighted mean size, and the particlesper mL, versus the total length of the shaking path, in cm.

FIG. 17 is a chart showing the relationship between the shakingfrequency, in RPM, and the total length of the shaking path, in cm, usedto obtain the test results shown in FIG. 16.

FIGS. 18(a)-(c) are charts showing the percentage of the vesicles havinga size less than 10 μm, the number or 20 weighted mean size, and theparticles per mL for three different types of shaking devices.

DESCRIPTION OF THE PREFERRED EMBODIMENT

According to the method of the current invention, vesicles of optimalsize are made by first placing an aqueous suspension 34, preferablycomprising lipids, into a container 9, as shown in FIG. 1.

As used herein, the term "vesicle" refers to a spherical entity which ischaracterized by the presence of an internal void. Preferred vesiclesare formulated from lipids, including the various lipids describedherein. In any given vesicle, the lipids may be in the form of amonolayer or bilayer, and the mono- or bilayer lipids may be used toform one or more mono- or bilayers. In the case of more than one mono-or bilayer, the mono- or bilayers are generally concentric. The vesiclesdescribed herein are also sometimes referred to as bubbles ormicrobubbles and include such entities commonly referred to as liposomesand micelles, and the like. Thus, the lipids may be used to form aunilamellar vesicle (comprised of one monolayer or bilayer), anoligolamellar vesicle (comprised of about two or about three monolayersor bilayers) or a multilamellar vesicle (comprised of more than aboutthree monolayers or bilayers). The internal void of the vesicles may befilled with a liquid, including, for example, an aqueous liquid, a gas,a gaseous precursor, and/or a solid or solute material, including, forexample, a targeting ligand and/or a bioactive agent, as desired.

"Liposome" refers to a generally spherical cluster or aggregate ofamphipathic compounds, including lipid compounds, typically in the formof one or more concentric layers. Most preferably the gas filledliposome is constructed of a single layer (i.e. unilamellar) or a singlemonolayer of lipid. A wide variety of lipids may be used to fabricatethe liposomes including phospholipids and non-ionic surfactants (e.g.niosomes). Most preferably the lipids comprising the gas filledliposomes are in the gel state at physiological temperature. Theliposomes may be cross-linked or polymerized and may bear polymers suchas polyethylene glycol on their surfaces. Targeting ligands directed toendothelial cells are bound to the surface of the gas filled liposomes.A targeting ligand is a substance which is bound to a vesicle anddirects the vesicle to a particular cell type such as and not limited toendothelial tissue and/or cells. The targeting ligand may be bound tothe vesicle by covalent or non-covalent bonds. The liposomes may also bereferred to herein as lipid vesicles. Most preferably the liposomes aresubstantially devoid of water in their interiors.

"Micelle" refers to colloidal entities which form from lipidic compoundswhen the concentration of the lipidic compounds, such as lauryl sulfate,is above a critical concentration. Since many of the compounds whichform micelles also have surfactant properties (i.e. ability to lowersurface tension and both water and fat loving--hydrophilic andlipophilic domains), these same materials may also be used to stabilizebubbles. In general these micellular materials prefer to adopt amonolayer or hexagonal H2 phase configuration, yet may also adopt abilayer configuration. When a micellular material is used to form a gasfilled vesicle, the compounds will generally adopt a radialconfiguration with the aliphatic (fat loving) moieties oriented towardthe vesicle and the hydrophilic domains oriented away from the vesiclesurface. For targeting to endothelial cells, the targeting ligands maybe attached to the micellular compounds or to amphipathic materialsadmixed with the micellular compounds. Alternatively, targeting ligandsmay be adsorbed to the surface of the micellular materials stabilizingthe vesicles.

A gas phase is employed above the aqueous suspension phase 34 in theremaining portion, or headspace 32, of the container 9. The introductionof the gas phase can be accomplished by purging the container 9 with agas, if a gas other than air is to be used for the gas phase, so thatthe gas occupies the headspace 32 above the aqueous suspension 34. Thus,prior to shaking, the container 9 contains an aqueous suspension phaseand a gaseous phase. The container 9 is then installed on the shaker arm7 of the shaking device 1 of the current invention, a preferredembodiment of which is shown in FIGS. 2, 3 and 6-11, and shaken for aperiod of time sufficient to form the desired vesicles.

Although filters may be used to further refine the size distribution ofthe vesicles after shaking, the focus of the current invention is on thecontrol of the shaking parameters in order to produce vesicles ofoptimal size prior to any post-shaking filtration. Toward this end, theinventors have found that the size of the vesicles produced by shakingis primarily a function of four variables:

(i) the composition of the aqueous suspension phase,

(ii) the composition of the gas phase in the headspace,

(iii) the volume of the container and the relative volume of theheadspace that is initially occupied by the gaseous phase, and

(iv) the definition of the primary shaking parameters--i.e., the shapeof the path traveled by the container during the shaking, the amplitudeof the shaking motion, and the duration and frequency of the shaking.

According to the method of the current invention, each of thesevariables should be adjusted in a process for making vesicles so as toobtain a desirable vesicle size distribution and concentration, with apreferable vesicle size distribution being one in which the vesicleshave a mean size of at least about 0.5 μm and in which at least 95% ofthe vesicles, and more preferably at least 99% of the vesicles, have adiameter less than 10 μm, and the concentration of vesicles produced isat least 100×10⁶ vesicles per mL and, more preferably, at least 500×10⁶vesicles per mL. Consequently, in sections I-IV, below, each of thesefour variables is discussed individually. In section V, a preferredapparatus for practicing the method of the current invention isdisclosed. Section VI discusses some applications of the vesicles madeaccording to the current invention.

I. The Composition of the Aqueous Suspension Phase

A wide variety of bubble coating agents may be employed in the aqueoussuspension phase. Preferably, the coating agents are lipids. The lipidsmay be saturated or unsaturated, and may be in linear or branched form,as desired. Such lipids may comprise, for example, fatty acids moleculesthat contain a wide range of carbon atoms, preferably between about 12carbon atoms and about 22 carbon atoms. Hydrocarbon groups consisting ofisoprenoid units, prenyl groups, and/or sterol moieties (e.g.,cholesterol, cholesterol sulfate, and analogs thereof) may be employedas well. The lipids may also bear polymer chains, such as theamphipathic polymers polyethyleneglycol (PEG) or polyvinylpyrrolidone(PVP) or derivatives thereof (for in vivo targeting), or charged aminoacids such as polylysine or polyarginine (for binding of a negativelycharged compound), or carbohydrates (for in vivo targeting) such as isdescribed in U.S. Pat. No. 4,310,505, or glycolipids (for in vivotargeting), or antibodies and other peptides and proteins (for in vivotargeting), etc., as desired. Such targeting or binding compounds may besimply added to the aqueous lipid suspension phase or may bespecifically chemically attached to the lipids. The lipids may also beanionic or cationic lipids, if desired, so that they may themselves becapable of binding other compounds such as pharmaceuticals, geneticmaterial, or other therapeutics.

Examples of classes of suitable lipids and specific suitable lipidsinclude: phosphatidylcholines, such as diolecylphosphatidylcholine,dimyristoylphosphatidylcholine, dipalmitoylphosphatidylcholine (DPPC),and distearoylphosphatidylcholine; phosphatidylethanolamines, such asdipalmitoylphosphatidylethanolamine (DPPE),dioleoylphosphatidylethanolamine andN-succinyl-dioleoylphosphatidylethanolamine; phosphatidylserines;phosphatidyl-glycerols; sphingolipids; glycolipids, such as gangliosideGM1; glucolipids; sulfatides; glycosphingolipids; phosphatidic acids,such as dipalmatoylphosphatidic acid (DPPA); palmitic fatty acids;stearic fatty acids; arachidonic fatty acids; lauric fatty acids;myristic fatty acids; lauroleic fatty acids; physeteric fatty acids;myristoleic fatty acids; palmitoleic fatty acids; petroselinic fattyacids; oleic fatty acids; isolauric fatty acids; isomyristic fattyacids; isopalmitic fatty acids; isostearic fatty acids; cholesterol andcholesterol derivatives, such as cholesterol hemisuccinate, cholesterolsulfate, and cholesteryl-(4'-trimethylammonio)-butanoate;

polyoxyethylene fatty acid esters; polyoxyethylene fatty acid alcohols;polyoxyethylene fatty acid alcohol ethers; polyoxyethylated sorbitanfatty acid esters; glycerol polyethylene glycol oxystearate; glycerolpolyethylene glycol ricinoleate; ethoxylated soybean sterols;

ethoxylated castor oil; polyoxyethylene-polyoxypropylene fatty acidpolymers; polyoxyethylene fatty acid stearates;12-(((7'-diethylaminocoumarin-3-yl)-carbonyl)-methylamino)-octadecanoicacid;N-[12-(((7'-diethylamino-coumarin-3-yl)-carbonyl)-methyl-amino)octadecanoyl]-2-amino-palmiticacid; 1,2-dioleoyl-sn-glycerol; 1,2-dipalmitoyl-sn-3-succinylglycerol;1,3-dipalmitoyl-2-succinyl-glycerol; and1-hexadecyl-2-palmitoyl-glycerophosphoethanolamine andpalmitoylhomocysteine; lauryltrimethylammonium bromide(lauryl-=dodecyl-); cetyltrimethylammonium bromide (cetryl-=hexadecyl-);myristyltrimethylammonium bromide (myristyl-=tetradecyl-);alkyldimethylbenzylammonium chlorides, such as wherein alkyl is a C₁₂,C₁₄ or C₁₆ alkyl; benzyldimethyldodecylammonium bromide;benzyldimethyldodecylammonium chloride, benzyldimethylhexadecylammoniumbromide; benzyldimethylhexadecylammonium chloride;benzyldimethyltetradecylammonium bromide;benzyldimethyltetradecylammonium chloride; cetyldimethylethylammoniumbromide; cetyldimethylethylammonium chloride; cetylpyridinium bromide;cetylpyridinium chloride;N-[1-2,3-dioleoyloxy)-propyl]-N,N,N-trimethylammonium chloride (DOTMA);1,2-dioleoyloxy-3-(trimethylammonio)propane (DOTAP); and1,2-dioleoyl-e-(4'-trimethylammonio)-butanoyl-sn-glycerol (DOTB).

As will be apparent to those skilled in the art, once armed with thepresent disclosures, the foregoing list of lipids is exemplary only, andother useful lipids, fatty acids and derivatives and combinationsthereof, may be employed, and such additional compounds are alsointended to be within the scope of the term lipid, as used herein. Asthe skilled artisan will recognize, such lipids and/or combinationsthereof may, upon shaking of the container, form liposomes (that is,lipid spheres having an internal void) which entrap gas from the gaseousphase in their internal void. The liposomes may be comprised of a singlelipid layer (a lipid monolayer), two lipid layers (a lipid bilayer) ormore than two lipid layers (a lipid multilayer).

As a general matter, it is preferred that the lipids remain in the gelstate, that is, below the gel state to liquid crystalline state phasetransition temperature (T_(m)) of the lipid material, particularlyduring shaking. Gel state to liquid crystalline state phase transitiontemperatures of various lipids are well known. Such temperatures mayalso be readily calculated using well known techniques. Table 1, below,from Derek Marsh, "CRC Handbook of Lipid Bilayers", page 139, CRC Press,Boca Raton, Fla. (1990), shows, for example, the main chain phasetransition temperatures for a variety of representative saturatedphosphocholine lipids.

                  TABLE 1                                                         ______________________________________                                        Saturated Diacyl-sn-Glycero-(3)-Phosphocholines:                              Main Chain Melting Transitions                                                                Main Phase                                                    # Carbons in Acyl                                                                             Transition                                                    Chains          Temperature ° C.                                       ______________________________________                                        1,2-(12:0)      -1.0                                                          1,2-(13:0)      13.7                                                          1,2-(14:0)      23.5                                                          1,2-(15:0)      34.5                                                          1,2-(16:0)      41.4                                                          1,2-(17:0)      48.2                                                          1,2-(18:0)      55.1                                                          1,2-(19:0)      61.8                                                          1,2-(20:0)      64.5                                                          1,2-(21:0)      71.1                                                          1,2-(22:0)      74.0                                                          1,2-(23:0)      79.5                                                          1,2-(24:0)      80.1                                                          ______________________________________                                    

In a preferred embodiment of the invention, the aqueous lipid phasefurther comprises a polymer, preferably an amphipathic polymer, andpreferably one that is directly bound (i.e., chemically attached) to thelipid. Preferably, the amphipathic polymer is polyethylene glycol or aderivative thereof. The most preferred combination is the lipiddipalmitoylphosphatidylethanolamine (DPPE) bound to polyethylene glycol(PEG), especially PEG of an average molecular weight of about 5000(DPPE-PEG5000). The PEG or other polymer may be bound to the DPPE orother lipid through a covalent linkage, such as through an amide,carbamate or amine linkage. Alternatively, ester, ether, thioester,thioamide or disulfide (thioester) linkages may be used with the PEG orother polymer to bind the polymer to, for example, cholesterol or otherphospholiplds. A particularly preferred combination of lipids is DPPC,DPPE-PEG5000 and DPPA, especially in a ratio of about 82%:8%:10% (mole%), DPPC: DPPE-PEG5000:DPPA.

Other coating agents that may alternatively, or in addition, be employedin the aqueous suspension phase include polymers such as proteins,natural and seminatural carbohydrates and synthetic polymers. A varietyof different proteins might be used in the invention to produce the gasfilled vesicles. Such proteins include albumin from natural (human andanimal) and recombinant origins, fibrin, collagen, antibodies andelastin. Natural polysaccharides include starch, cellulose, alginicacid, pectin, dextran, heparin and hyaluronic acid. Semi-naturalpolysaccharides include methylcellulose, hydroxypropylcellulose,carboxmthylycellulose and hydroxyethyl starch. Synthetic polymersinclude polyvinylpyrrolidone, copolymers of ethylene and propyleneglycol (e.g. Pluronic F-68 and the other Pluronics), polyethyleneglycol,polyvinylalcohol, polylactic acid, copolymers of lactic and glycolicacids, polymethacrylate and double ester polymers. Also inorganic mediasuch as hydroxyapatite and calcium pyrophosphate may be used in theinvention. In all these cases the bubble coating agents are suspended inthe aqueous phase in a container with a head space of the preselectedgas and then shaken. This results in formation of the stabilized, coatedvesicles. As one skilled in the art would recognize, once armed with thedisclosure of this invention, a wide variety of different stabilizingagents can be used to make vesicles according to the principles of theinvention.

In one experiment with human serum albumin, BRL-Life Technologies,Gaithersburg, Md., a 10 ml glass vial containing an albumin solution anda head space of perfluropropane gas (vol of liquid=6 ml, 5 mg per mlalbumin solution) was shaken for 2 minutes at 2800 RPM with a Wig-L-Bug™to produce albumin coated perfluoropropane vesicles having a meandiameter of 5 microns, with a concentration of 50 million particles perml.

In addition, the use of the invention is compatible with a variety ofsuspending and/or viscosity agents. The phrase suspending agent, as usedherein, denotes a compound that assists in providing relative uniformityor homogeneity to the contrast medium. A number of such agents areavailable, including xanthan gum, acacia, agar, alginic acid, aluminummonostearate, bassorin, karaya, gum arabic, unpurified bentonite,purified bentonite, bentonite magma, carbomer 934P, calciumcarboxymethylcellulose, sodium carboxymethylcellulose,carboxymethylcellulose sodium 12, carrageenan, cellulose(microcrystalline), dextran, gelatin, guar gum, hydroxyethylcellulose,hydroxypropylcellulose, hydroxypropylmethylcellulose, magnesium aluminumsilicate, methylcellulose, pectin, casein, gelatin, polyethylene oxide,polyvinyl alcohol, povidone, propylene glycol, alginate, silicondioxide, silicon dioxide colloidal, sodium alginate and other alginates,and tragacanth. As those skilled in the art would recognize, wide rangesof suspending agent can be employed in the contrast medium of theinvention, as needed or desired.

The concentrations of these agents will vary depending upon the bubblestabilizing media which are selected and the shaking parameters may alsovary depending upon the materials employed. Lipids, because of theirbiocompatibility, low toxicity, availability as pure and pharmaceuticalgrade materials are the preferred bubble coating agents to make the gasfilled vesicles of this invention.

To prepare the aqueous phase, the lipids, or other coating agent, may becombined with water (preferably distilled water), normal (physiological)saline solution, phosphate buffered saline solution, or other aqueousbased solution, as will be apparent to those skilled in the art.

As one skilled in the art would recognize, once armed with the substanceof the present disclosure, various additives may be employed in theaqueous suspension phase of the invention to stabilize that phase, or tostabilize the gas-filled vesicles upon shaking. If desired, theseadditives may be added to the aqueous suspension phase prior to shaking,or may be added to the composition after shaking and resultantpreparation of the gas-filled vesicles. The use of such additives will,of course, be dependent upon the particular application intended for theresultant gas-filled vesicles, as will be readily apparent to thoseskilled in the art.

A number of stabilizing agents which may be employed in the presentinvention are available, including xanthan gum, acacia, agar, agarose,alginic acid, alginate, sodium alginate, carrageenan, dextran, dextrin,gelatin, guar gum, tragacanth, locust bean, bassorin, karaya, gumarabic, pectin, casein, bentonite, unpurified bentonite, purifiedbentonite, bentonite magma, colloidal, cellulose, cellulose(microcrystalline), methylcellulose, hydroxyethylcellulose,hydroxypropylcellulose, hydroxypropylmethylcellulose,carboxymethylcellulose, calcium carboxymethylcellulose, sodiumcarboxymethylcellulose, carboxymethylcellulose sodium 12, as well asother natural or modified natural celluloses, polysorbate, carbomer934P, magnesium aluminum silicate, aluminum monostearate, polyethyleneoxide, polyvinylalcohol, povidone, polyethylene glycol, propyleneglycol, polyvinylpyrrolidone, silicon dioxide, silicon dioxidecolloidal.

Also, compounds such as such as perfluorooctylbromide (PFOB),perfluorooctyliodide, perfluorotripropylamine, andperfluorotributylamine may be utilized in the lipid phase as stabilizingagents. Perfluorocarbons with greater than five carbon atoms willgenerally be liquid at body temperature, and such perfluorocarbons arealso highly preferred as stabilizing agents. Suitable perfluorcarbonsinclude perfluorohexane, perfluoroheptane, perfluorooctane,perfluorodecalin, and perfluorododecalin. In addition, perfluorinatedlipids or partially fluorinated lipids may be used as well to help instabilization. As will be apparent to those skilled in the art, a widevariety of perfiluorinated and partially fluorinated analogs of thelipids described in the present invention may be used. Because of theirrelative hydrophobic nature with respect to the hydrocarbon lipids, suchperfluorinated or partially fluorinated lipids may even provideadvantages in terms of stability. Examples of perfluorinated orpartially fluorinated lipids are F₆ C₁₁ phosphatidylcholine(PC) and F₈C₅ PC. Such analogs are described, for example, in Santaella et al.,Federation of European Biochemical Societies (FEBS), Vol. 336, No. 3,pp. 418-484 (1993), the disclosures of which are hereby incorporatedherein by reference in their entirety.

A wide variety of biocompatible oils may also be used for the purpose ofassisting stabilization, such as peanut oil, canola oil, olive oil,saffower oil, corn oil, almond oil, cottonseed oil, persic oil, sesameoil, soybean oil, mineral oil, mineral oil light, ethyl oleate, myristylalcohol, isopropyl myristate, isopropyl palmitate, octyldodecanol,propylene glycol, glycerol, squalene, or any other oil commonly known tobe ingestible. These may also include lecithin, sphingomyelin,cholesterol, cholesterol sulfate, and triglycerides.

Stabilization may also be effected by the addition of a wide variety ofviscosity modifiers (i.e., viscosity modifying agents), which may serveas stabilizing agents in accordance with the present invention. Thisclass of compounds include but are by no means restricted to: 1)carbohydrates and their phosphorylated and sulfonated derivatives; 2)polyethers with molecular weight ranges between 400 and 8000; 3) di- andtrihydroxy alkanes and their polymers in the molecular weight rangebetween 800 and 8000. Liposomes may also be used in conjunction withemulsifying and/or solubilizing agents which may consist of, but are byno means limited to, acacia, cholesterol, diethanolamine, glycerylmonostearate, lanolin alcohols, lecithin, mono- and diglycerides,monoethanolamine, oleic acid, oleyl alcohol, poloxamer, polyoxyethylene50 stearate, polyoxyl 35 castor oil, polyoxyl 10 oleyl ether, polyoxyl20 cetostearyl ether, polyoxyl 40 stearate, polysorbate 20, polysorbate40, polysorbate 60, polysorbate 80, propylene glycol diacetate,propylene glycol monostearate, sodium lauryl sulfate, sodium stearate,sorbitan monolaurate, sorbitan monooleate, sorbitan monopalmitate,sorbitan monostearate, stearic acid, trolamine, emulsifying wax,Pluronic F61, Pluronic F64 and Pluronic F68.

Other agents which may be added include tonicity agents such aspolyalcohols such as glycerol, propylene glycol, polyvinylalcohol,polyethyeneglycol, glucose, mannitol, sorbitol, sodium chloride and thelike.

If desired, anti-bactericidal agents and/or preservatives may beincluded in the formulation. Such agents include sodium benzoate, allquaternary ammonium salts, sodium azide, methyl paraben, propyl paraben,sorbic acid, potassium sorbate, sodium sorbate, ascorbylpalmitate,butylated hydroxyanisole, butylated hydroxytoluene, chlorobutanol,dehydroacetic acid, ethylenediamine tetraacetic acid (EDTA),monothioglycerol, potassium benzoate, potassium metabisulfite, potassiumsorbate, sodium bisulfite, sulfur dioxide, and organic mercurial salts.

If desired, an osmolarity agent may be utilized to control theosmolarity. Suitable osmotically active materials include suchphysiologically compatible compounds as monosaccharide sugars,disaccharide sugars, sugar alcohols, amino acids, and various syntheticcompounds. Suitable monosaccharide sugars or sugar alcohols include, forexample, erythrose, threose, ribose, arabinose, xylose, lyxose, allose,altrose, glucose, mannose, idose, galactose, talose, trehalose,ribulose, fructose, sorbitol, mannitol, and sedoheptulose, withpreferable monosaccharides being fructose, mannose, xylose, arabinose,mannitol and sorbitol. Suitable disaccharide sugars include, forexample, lactose, sucrose, maltose, and cellobiose. Suitable amino acidsinclude, for example, glycine, serine, threonine, cysteine, tyrosine,asparagine, glutamine, aspartic acid, glutamic acid, lysine, arginineand histidine. Synthetic compounds include, for example, glycerol,propylene glycol, polypropylene glycol, ethylene glycol, polyethyleneglycol and polyvinyl-pyrrolidone. Various other suitable osmoticallyactive materials are well known to those skilled in the art, and areintended to be within the scope of the term osmotically active agent asused herein.

A variety of polymers, such as those discussed above, may also be addedfor a variety of different purposes and uses.

As those skilled in the art would recognize, a wide range of additiveamounts, such as the suspending agents described above, may be employedin the aqueous suspension phase of the invention, as needed or desired,depending upon the particular end use. Such additives generally maycomprise from between 0.01% by volume to about 95% by volume of theresultant contrast agent formulation, although higher or lower amountsmay be employed. By way of general guidance, a suspending agent istypically present in an amount of at least about 0.5% by volume, morepreferably at least about 1% by volume, even more preferably at leastabout 10% by volume. Generally the suspending agent is typically presentin an amount less than about 50% by volume, more preferably less thanabout 40% by volume, even more preferably less than about 30% by volume.A typical amount of suspending agent might be about 20% by volume, forexample. Also, typically, to achieve generally preferred ranges ofosmolarity, less than about 25 g/l, more preferably less than about 20g/l, even more preferably less than about 15 g/l, and still morepreferably less than about 10 g/l of the osmotically active materialsare employed, and in some instances no osmotically active materials areemployed. A most preferred range of osmotically active materials isgenerally between about 0.002 g/l and about 10 g/l. These, as well asother, suitable ranges of additives will be readily apparent to thoseskilled in the art, once placed in possession of the present invention.

A wide variety of therapeutic and/or diagnostic agents may also beincorporated into the aqueous suspension phase simply by adding thedesired therapeutic or diagnostic agents to that phase. Suitabletherapeutic and diagnostic agents, and suitable amounts thereof, will bereadily apparent to those skilled in the art, once armed with thepresent disclosure. These agents may be incorporated into or onto lipidmembranes, or encapsulated in the resultant liposomes.

To further improve the magnetic effect of the resultant gas-filledvesicles for MRI, for example, one or more MRI contrast enhancingagents, such as paramagnetic or superparamagnetic contrast enhancingagents, may be added. Useful MRI contrast enhancing agents includeparamagnetic ions such as transition metals, including iron (Fe⁺³),copper (Cu⁺²), and manganese (Mn⁺²) and the lanthanides such asgadolinium (Gd⁺³) and dysprosium (Dy⁺³), nitroxides, iron oxides (Fe₃O₄), iron sulfides and paramagnetic particles such as manganese (Mn⁺²)substituted hydroxyapatites. As well, agents such as chromium (Cr⁺³),nickel (Ni⁺²), cobalt (Co⁺²) and euronium (Eu⁺²) are other examples ofparamagnetic ions that may be used. Other contrast enhancing agents suchas nitroxide radicals or any other atom that maintains an unpairedelectron spin with paramagnetic properties may be used. Ideally, thecontrast enhancing agent is added to the aqueous suspension phase priorto shaking, and is designed such that after shaking, the contrastenhancing agent is incorporated into or onto the surface of theresultant gas-filled vesicles, although addition after vesiclepreparation is also possible. The resulting gas-filled vesicles may havegreatly enhanced relaxivity, providing an especially effective contrastagent for magnetic resonance imaging. By way of example, manganese(Mn⁺²) will incorporate itself onto the head groups of the lipid whenphosphatidylcholine or phosphatidylserine is used in the aqueous lipidphase. If desired, the metals may be chelated using liposolublecompounds as shown, for example, in Unger et al., U.S. Pat. No.5,312,617, the disclosure of which is hereby incorporated herein byreference in its entirety. Such liposoluble compounds are quite useful,as they will readily incorporate into the liposome membrane. Iron oxidesand other particles should generally be small, preferably less thanabout 1μ, more preferably less than about 200 nm, and most preferablyless than 100 nm, to achieve optimal incorporation into or onto theliposome surface. For improved incorporation, iron oxides coated withaliphatic or lypophyllic compounds may be used as these will tend toincorporate themselves into the lipid coating of the bubble surface.

It also is within the realm of the present invention that the aqueoussuspension phase may contain an ingredient to cause gelation, such as aningredient that will cause gelation with lipid polymers and metals whichdo not spontaneously gel, or that will enhance gelation. Gelling agentssuch as polyvalent metal cations, sugars and polyalcohols may beemployed. Exemplary polyvalent metal cations useful as gelling agentsinclude calcium, zinc, manganese, iron and magnesium. Useful sugarsinclude monosaccharides such as glucose, galactose, fructose, arabinose,allose and altrose, disaccharides such as maltose, sucrose, cellobioseand lactose, and polysaccharides such as starch. Preferably, the sugaris a simple sugar, that is, monosaccharide or a disaccharide.Polyalcohol gelling agents useful in the present invention include, forexample, glycidol, inositol, mannitol, sorbitol, pentaerythritol,galacitol and polyvinylalcohol. Most preferably, the gelling agentemployed in the present invention is sucrose and/or calcium. Theparticular gelling agents which may be employed in the variousformulations of the present invention will be readily apparent to oneskilled in the art, once armed with the present disclosure.

Combinations of lipids, e.g. phosphatidic acid with calcium or magnesiumsalts and polymers such as alginic acid, hyaluronic acid orcarboxymethyl cellulose may be used to stabilize lipids. It ishypothesized that the divalent cations form metal bridges between thelipids and polymers to stabilize the gas-filled liposomes within thelipid/polymeric systems. Similarly, suspensions containing mixtures ofchitosan (or chitin-based materials), polylysine, polyethyleneimine andalginic acid (or its derivatives) or hyaluronic acid may be prepared.

It has been discovered that the different materials within the aqueousphase may be important in controlling resultant gas-filled vesicle size.Table 2 shows the sizes of liposomes produced by shaking sterilecontainers filled with an aqueous phase and a headspace of nitrogen. Inall cases, the liposome size was measured by a Particle Sizing SystemModel 770 light obscuration particle sizer (Particle Sizing Systems,Santa Barbara, Calif.). As the data reveals, the ratio of lipids in theaqueous phase affects the size distribution of the resulting gas-filledliposomes. Specifically, Table 2 below shows the effect of lipidcomposition on the average liposome size.

                  TABLE 2                                                         ______________________________________                                        Effect of Lipid Composition on Average Liposome Size                          Lipid Composition*                                                                           Average Liposome Size                                          ______________________________________                                        77.5:15:7.5    5.26 μm                                                     77.5:20:2.5    7.33 μm                                                     82:10:8        6.02 μm                                                     ______________________________________                                         *Ratios of dipalmitoylphosphatidylcholine:dipalmitoylphosphatidic acid:       dipalmitoylphosphatidylethanolaminePEG5000, in mole %.                   

Table 3 demonstrates the dependence of the concentration of a definedlipid composition mixture upon the average liposome size. As shown inTable 3, variations in the total concentrations of lipid are alsoimportant in affecting liposome size after shaking. In these experimentsthe ratio of the three different lipid components was held constant andthe concentration of lipid was varied between 0.5 and 5.0 mg ml⁻¹ in theaqueous phase. The gas used was nitrogen. The optimal size vesicles forultrasonic diagnosis with a headspace of perfluorobutane was producedwhen the lipid concentration in the aqueous phase was 1.0 mg ml⁻¹.

                  TABLE 3                                                         ______________________________________                                        Effect of Lipid Concentration on Average Liposome Size                        Lipid Concentration*                                                                         Average Liposome Size                                          ______________________________________                                        1 mg ml.sup.-1 1.8 μm                                                      3 mg ml.sup.-1 4.0 μm                                                      5 mg ml.sup.-1 7.2 μm                                                      ______________________________________                                         *Lipid concentration forall samples was based upon a mole % ratio of          dipalmitpylphosphatidylcholine:dipalmitoylphosphatidic                        acid:dipalmitoylphosphatidylethanolaminePEG5000 of 82:10:8. The gas used      was nitrogen.                                                            

The size of vesicles may also depend on the concentration of stabilizingmedia, e.g. lipids. For example it has been discovered that a 1.0 mgml⁻¹ lipid concentration produces gas-filled liposomes of about the samediameter when nitrogen is used, as the 5.0 mg ml⁻¹ concentration oflipids with perfluorobutane. However, it has been found that the higherconcentration may result in a distribution skewed a bit more towardslarger gas-filled liposomes. This phenomenon tends to reflect theincreased stability of the gas-filled liposomes at higher lipidconcentration. It is therefore believed that the higher concentration oflipid either contributes to the stability by acting as a stabilizingagent in the aqueous phase or, the higher lipid concentration providesmore lamellae around the gas, making them more stable, and thus allowinga greater proportion of the larger liposomes to persist.

It is also believed that the surface tension at the gas-filled vesicleinterface and the aqueous milieu is an additional determining factor inthe ultimate size of the gas-filled vesicle, when taken into accountalong with the other variables.

II. The Composition of the Gaseous Phase

A wide variety of different gases may be employed in the gaseous phaseof the present invention. Preferably the gases are substantiallyinsoluble in the aqueous suspension phase. By substantially insoluble,it is meant that the gas maintains a solubility in water at 20° C. and 1atmosphere of pressure of equal to or less than about 18 ml of gas perkg of water. As such, substantially insoluble gases have a solubilitywhich is less than the solubility of nitrogen gas. Preferably, thesolubility is equal to or less than about 15 ml of gas per kg of water,more preferably equal to or less than about 10 ml of gas per kg ofwater, at 20° C. and 1 atmosphere of pressure. In one preferable classof gases, the solubility is between about 0.001 and about 18 ml of gasper kg of water, or between about 0.01 and about 15 ml of gas per kg ofwater, or between about 0.1 and about 10 ml of gas per kg of water, orbetween about 1 and about 8 ml of gas per kg of water, or between about2 and 6 ml per kg of water, at the aforementioned temperature andpressure. Perfluorocarbon gases and the fluorinated gas sulfurhexafluoride are, for example, less soluble than 10 ml of gas per kg ofwater, at 20° C. and 1 atmosphere of pressure, and thus are preferred.Gases which are not substantially insoluble, as defined herein, arereferred to as soluble gases.

Other suitable substantially insoluble or soluble gases include, but arenot limited to, hexafluoroacetone, isopropylacetylene, allene,tetrafluoroallene, boron trifluoride, 1,2-butadiene, 1,3-butadiene,1,2,3-trichlorobutadiene, 2-fluoro-1,3-butadiene, 2-methyl-1,3butadiene, hexafluoro-1,3-butadiene, butadiyne, 1-fluorobutane,2-methylbutane, decafluorobutane (perfluorobutane), decafluoroisobutane(perfluoroisobutane), 1-butene, 2-butene, 2-methy-1-butene,3-methyl-1-butene, perfluoro-1-butene, perfluoro-1-butene,perfluoro-2-butene, 4-phenyl-3-butene-2-one, 2-methyl-1-butene-3-yne,butylnitrate, 1-butyne, 2-butyne,2-chloro-1,1,1,4,4,4-hexafluoro-butyne, 3-methyl-1-butyne,perfluoro-2-butyne, 2-bromo-butyraldehyde, carbonyl sulfide,crotononitrile, cyclobutane, methylcyclobutane, octafluorocyclobutane(perfluorocyclobutane), perfluoroisobutane, 3-chlorocyclopentene,cyclopropane, 1,2-dimethylcyclopropane, 1,1-dimethylcyclopropane, ethylcyclopropane, methylcyclopropane, diacetylene,3-ethyl-3-methyldiaziridine, 1,1,1-trifluorodiazoethane, dimethylamine,hexafluorodimethylamine, dimethylethylamine, bis-(dimethylphosphine)amine, 2,3-dimethyl-2-norbornane, perfluoro-dimethylamine,dimethyloxonium chloride, 1,3-dioxolane-2-one,1,1,1,1,2-tetrafluoroethane, 1,1,1-trifluoroethane,1,1,2,2-tetrafluoroethane, 1,1,2-trichloro-1,2,2-trifluoroethane,1,1-dichloroethane, 1,1-dichloro-1,2,2,2-tetrafluoroethane,1,2-difluoroethane, 1-chloro-1,1,2,2,2-pentafluoroethane,2-chloro-1,1-difluoroethane, 1-chloro-1,1,2,2-tetrafluoro-ethane,2-chloro-1,1-difluoroethane, chloroethane, chloropentafluoroethane,dichlorotrifluoroethane, fluoroethane, nitropentafluoroethane,nitrosopentafluoro-ethane, perfluoroethane, perfluoroethylamine, ethylvinyl ether, 1,1-dichloroethylene, 1,1-dichloro-1,2-difluoro-ethylene,1,2-difluoroethylene, methane, methane-sulfonyl-chlori-detrifluoro,methane-sulfonyl-fluoride-trifluoro, methane-(pentafluorothio)trifluoro,methane-bromo-difluoro-nitroso, methane-bromo-fluoro,methane-bromo-chloro-fluoro, methane-bromo-trifluoro,methane-chloro-difluoro-nitro, methane-chloro-dinitro,methane-chloro-fluoro, methane-chloro-trifluoro,methane-chloro-difluoro, methane-dibromo-difluoro,methane-dichloro-difluoro, methane-dichloro-fluoro, methane-difluoro,methane-difluoro-iodo, methane-disilano, methane-fluoro,methane-iodomethane-iodo-trifluoro, methane-nitro-trifluoro,methane-nitroso-triofluoro, methane-tetrafluoro,methane-trichloro-fluoro, methane-trifluoro,methanesulfenylchloride-trifluoro, 2-methyl butane, methyl ether, methylisopropyl ether, methyl lactate, methyl nitrite, methyl sulfide, methylvinyl ether, neopentane, nitrogen (N₂), nitrous oxide, 1,2,3-nonadecanetricarboxylic acid-2-hydroxycrimethylester, 1-nonene-3-yne, oxygen (O₂),oxygen 17 (¹⁷ O₂), 1,4-pentadiene, n-pentane, dodecafluoropentane(perfluoropentane), tetradecafluorohexane (perfluorohexane),perfluoroisopentane, perfluoroneopentane, 2-pentanone-4-amino-4-methyl,1-pentene, 2-pentene {cis}, 2-pentene {trans}, 1-pentene-3-bromo,1-pentene-perfluoro, phthalic acid-tetrachloro,piperidine-2,3,6-trimethyl, propane, propane-1,1,1,2,2,3-hexafluoro,propane-1,2-epoxy, propane-2,2 difluoro, propane-2-amino,propane-2-chloro, propane-heptafluoro-1-nitro,propane-heptafluoro-1-nitroso, perfluoropropane, propene,propyl-1,1,1,2,3,3-hexafluoro-2,3 dichloro, propylene-1-chloro,propylene-chloro-{trans}, propylene-2-chloro, propylene-3-fluoro,propylene-perfluoro, propyne, propyne-3,3,3-trifluoro, styrene-3-fluoro,sulfur hexafluoride, sulfur (di)-decafluoro(S₂ F₁₀),toluene-2,4-diamino, trifluoroacetonitrile, trifluoromethyl peroxide,trifluoromethyl sulfide, tungsten hexafluoride, vinyl acetylene, vinylether, neon, helium, krypton, xenon (especially rubidium enrichedhyperpolarized xenon gas), carbon dioxide, helium, and air. Fluorinatedgases (that is, a gas containing one or more fluorine molecules, such assulfur hexafluoride), fluorocarbon gases (that is, a fluorinated gaswhich is a fluorinated carbon or gas), and perfluorocarbon gases (thatis, a fluorocarbon gas which is fully fluorinated, such asperfluoropropane and perfluorbutane) are preferred.

While virtually any gas may be theoretically employed in the gaseousphase of the present invention, a particular gas may be chosen tooptimize the desired properties of the resultant contrast medium and tofit the particular diagnostic application. It has been found, forexample, that certain gases make more stable gas-filled vesicles uponshaking than other gases, and such gases are preferred. It has also beenfound that certain gases provide better imaging results on diagnosticimaging such as ultrasound or MRI.

As an example of increasing stability of the gas-filled vesicles, it hasbeen found that carbondioxide<oxygen<air<nitrogen<neon=helium<perfluorocarbon gases. Forthese, as well as other, reasons fluorinated gases, particularlyperfluorocarbon gases, are preferred.

Also, although in some cases soluble gases will function adequately asthe gaseous phase in the present invention, substantially insolublegases tend to result in greater stability than gases with highersolubility, particularly upon creation of the contrast agent on shaking.Also, it will be easier to keep a gaseous phase with such insolublegases substantially separate from the aqueous suspension phase prior toshaking, in accordance with the present invention. Thus, substantiallyinsoluble gases, as earlier defined, are preferred.

The quality of ultrasound images and the duration of such images alsocorrelates with the solubility of the gas in the aqueous milieu. Thedecrease in gas solubility, in general, offers a better resolved imageof longer duration on ultrasound.

Additionally, it has been generally observed that the size of agas-filled vesicles produced by shaking correlates with the solubilityof the gas in the aqueous milieu, with the gases of greater solubilityresulting in larger gas-filled vesicles.

It is also believed that the size of the vesicles may be influenced bythe interaction of the gas with the inner wall of the vesicles.Specifically, it is believed that the interaction at the interfaceaffects the tension and, consequently, the outward force of the interiorgas on the interior vesicle wall of the vesicle. A decrease in tensionallows for smaller vesicles by decreasing the force exerted by theinterior gas, thus allowing the force exerted on the exterior of thevesicle by the aqueous milieu to contract the gas-filled vesicle.

The solubility of gases in aqueous solvents may be estimated by the useof Henry's Law, since it is generally applicable to pressures up toabout 1 atmosphere pressure and for gases that are slightly soluble(Daniels, F. and Alberty, R. A., Physical Chemistry, 3rd Edition, Wiley& Sons, Inc., New York, 1966). As an example, oxygen has a solubility of31.6 ml per kg of water at 25° C., atmospheric air possesses asolubility of 21.36 ml in 1 kg of water at 25° C., nitrogen maintains asolubility of approximately 18.8 ml kg⁻¹ at 25° C. Sulfur hexafluoride,on the other hand, has a solubility of approximately 5.4 ml kg⁻¹ at 25°C.

In sum, the fluorinated gases, fluorocarbon gases, and perfluorcarbongases are preferred for reasons of stability, insolubility, andresultant vesicle size. Particularly preferred are the fluorinated gassulfur hexafluoride, and the perfluorocarbon gases perfluoropropane,perfluorobutane, perfluorocyclobutane, perfluoromethane,perfluoroethane, and perfluoropentane, especially perfluoropropane andperfluorobutane.

It should be noted that perfluorocarbons having less than five carbonatoms are gases at room temperature.

Perfluoropentane, for example, is a liquid until about 27° C. Above thistemperature it will occupy the headspace of the container. It has beendemonstrated that perfluoropentane also may be used to fill theheadspace (that is, the space in the vial above the lipid suspensionphase) even at room temperature, however. By selecting a defined valueof liquid perfluoropentane calculated to fill the headspace and addingthe liquid to the container at low temperature, e.g., -20° C., and thenevacuating the container (effectively removing the headspace of air) andthen sealing the container, perfluoropentane will undergo a transitionfrom liquid phase to vapor phase at a temperature lower than its boilingpoint at 1 atmosphere. Thus, at room temperature it will occupy some orall of the headspace with gas. As those skilled in the art willrecognize, one may estimate the decrease in the liquid phase to vaporphase transition temperature by using a common "rule of thumb" estimate.Specifically, for every decrease in pressure by half, the boilingtemperature will decrease by about 10° C. Alternatively, one maycalculate the decrease in temperature as a function of decreasedpressure by using relationships based upon the ideal gas law based uponBoyle's law. Another method for filling the headspace withperfluoropentane is to first evacuate the headspace and then to fill theheadspace with perfluoropentane gas above 27° C. Of course, this methodis not limited to perfluoropentane alone, but applies to allperfluorocarbon gases, as well as gases in general, provided the boilingpoint of the gas is known.

If desired, two or more different gases may be used together to fill theheadspace. A mixture of gases may have a number of advantages in a widevariety of applications of the resultant gas-filled vesicles (such asapplications in ultrasound imaging, MR imaging, etc.). It has been foundthat a small amount of a substantially insoluble gas may be mixed with asoluble gas to provide greater stability than would be expected by thecombination. For example, a small amount of perfluorocarbon gas(generally at least about 1 mole %, for example) may be mixed with air,nitrogen, oxygen, carbon dioxide or other more soluble gases. Theresulting gas-filled vesicle contrast agent produced post-shaking maythen be more stable than the air, nitrogen, oxygen, carbon dioxide orother more soluble gases alone.

Additionally, the use of a mixture of gases may be used to compensatefor the increase in gas-filled vesicle size which might otherwise occurin vivo were pure perfluorocarbon gas containing vesicles to be injectedin vivo. It has been found that some perfluorocarbon gases may tend toabsorb or imbibe other gases such as oxygen. Thus, if theperfluorocarbon gas is injected intravenously, it may take up the oxygenor other soluble gases dissolved in the circulating blood. The resultingvesicles may then grow in vivo as a result of this uptake. Armed with aknowledge of this phenomenon, one may then premix the perfluorocarbongas with a soluble gas, such as air, nitrogen, oxygen, carbon dioxide,thereby saturating the perfluorocarbon of its absorptive or imbibingproperties. Consequently, this would retard or even eliminate thepotential for expansion of the gas-filled vesicles in the bloodstream.This is significant in light of the fact that should a vesicle grow to asize greater than 10 μM, potentially dangerous embolic events may occurif administered in the bloodstream. By filling the headspace with moresoluble gases than perfluorocarbon gas, along with the perfluorocarbongas, the gas-filled vesicles will generally not undergo this increase insize after injection in vivo. Thus, as a result of the presentinvention, the problem of embolic events as a result of vesicleexpansion may be circumvented by producing vesicles where such expansionis eliminated or sufficiently retarded.

Thus, in accordance with the present invention, if desired, asubstantially insoluble gas may be combined with a soluble gas toefficiently produce highly effective and stable gas-filled vesicles.

Multiple samples of lipid solutions (1 mg per mL; 82:10:8 mole % ratiosof DPPC:DPPA:DPPE-PEG-5000) in 8:1:1 weight ratios of normalsaline:glycerol:proplyene glycol in 2 ml vials (actual size 3.7 ml)Wheaton Industries (Millville, N.J.) were placed on a modified EdwardsModel SO4 lyophilizer with four cubic foot capacity and subjected toreduced pressure. The headspaces of the vials, which formed 60% of thetotal volume, were then instilled with 80% PFP with 20% air, 60% PFPwith 40% air, 50% PFP with 50% air, 20% PFP with 80% air, or 100% air.The percentages of gas in the headspaces of the different samples wereconfirmed by gas chromatography a Hewlett Packard Gas ChromatographModel 1050L interfaced with Hewlett Packard Chem™ softward. The mode ofdetection was Flame ionization detection. The samples were then shakenat 3,300 RPM for 60 seconds using a standard Wig-L-Bug™ model 3110B andthe sizes and vesicles counts determined by optical particle sizing. AnOptical Particle Sizer (Particle Sizing Systems, Santa Barbara, Calif.)was used to analyze gas-filled vesicle size and total counts. A samplevolume of 5 microliters was used for each analysis, with four samplesused for each determination. The results are shown above in Table 4.

As shown in Table 4, even when only 20% of the gas was PFP (asubstantially insoluble gas) and 80% of the gas was air (a mixture ofsoluble gases), 100 fold more vesicles were produced than when air alone(0% PFP) was used. Moreover, when air alone (0% PFP) was used, thevesicles were much less stable and a larger fraction were above 10microns. The 20% PFP and 80% air vesicles, however, appeared just asstable as the 80% PFP and 20% air vesicles, as well as the otherintermediate PFP concentration samples, and the 20% PFP with 80% airproduced about as many gas-filled vesicles as 80% PFP with 20% air.

                                      TABLE 4                                     __________________________________________________________________________    Effect of Percent Perfluoropropane on Vesicle Size and                        Number                                                                                    Volume                                                                             Estimated                                                                           Percentage of                                                                        Estimated #                                                                         Percentage of                             Gas Number  Weighted                                                                           Number of                                                                           Particles                                                                            of Particles                                                                        Particles                                 % PFP                                                                             Weighted Mean                                                                         Mean Particles                                                                           <10 μm                                                                            per mL                                                                              >10 μm                                 __________________________________________________________________________    80%                                                                           Average                                                                           2.37    28.76                                                                               5.45E+05                                                                           98.94   1.10E+09                                                                            1.05                                     STDev                                                                             0.07     0.82                                                                               4.67E+04                                                                            0.08   8.20E+07                                                                            0.07                                     CV  3%       3%   9%    0%     7%    7%                                       60%                                                                           Average                                                                           2.14    20.75                                                                               5.87E+05                                                                           99.36   1.15E+09                                                                            0.64                                     STDev                                                                             0.02     5.93                                                                               7.08E+04                                                                            0.10   1.27E+08                                                                            0.09                                     CV  1%      29%  12%    0%    11%   14%                                       50%                                                                           Average                                                                           2.13    30.35                                                                               5.23E+05                                                                           99.29   1.07E+09                                                                            0.68                                     STDev                                                                             0.07    12.15                                                                               1.49E+04                                                                            0.11   4.37E+07                                                                            0.10                                     CV  3%      40%   3%    0%     4%   15%                                       20%                                                                           Average                                                                           2.00    13.64                                                                               5.35E+05                                                                           99.61   1.07E+09                                                                            0.41                                     STDev                                                                             0.04     6.79                                                                               2.26E+04                                                                            0.06   3.92E+07                                                                            0.07                                     CV  2%      50%   4%    0%     4%   16%                                       0%                                                                            Average                                                                           2.30    93.28                                                                               5.03E+03                                                                           98.23   1.00E+07                                                                            1.93                                     STDev                                                                             0.21    66.05                                                                               4.96E+02                                                                            0.26   8.60E+05                                                                            0.36                                     CV  9%      71%  10%    0%     9%   19%                                       __________________________________________________________________________

In Table 4, STDev=Standard Deviation, and CV=Coefficient of Variance.Also in Table 4, E+ denotes an exponent to a certain power, for example,5.45E+05=5.45×10⁵.

In short, it has been found that only a small amount of a relativelyinsoluble gas (such as PFP) is needed to stabilize the vesicles, withthe vast majority of the gas being a soluble gas. Although the effectivesolubility of the combination of two or more gases, as calculated by theformula below: ##EQU1## may be only slightly different than thesolubility of the soluble gas, there is still a high gas-filled vesiclecount and gas-filled vesicle stability with only a small amount ofinsoluble gas in added.

Although not intending to be bound by any theory of operation, it isbelieved that the substantially insoluble gas is important for amembrane stabilizing effect. Indeed, it is believed that thesubstantially insoluble gas (such as PFP) acts as a barrier against thelipid membrane, possibly effectively forming a layer on the innersurface of the membrane, which retards egress of the soluble gas (suchas air, nitrogen, etc.). This discovery is both surprising and useful,as this allows one to use only a small amount of the substantiallyinsoluble gas (e.g., a perfluorocarbon or other fluorinated gas) andprimarily a more biocompatible (less potentially toxic) gas such as airor nitrogen to comprise most of the vesicle volume.

The amount of substantially insoluble gases and soluble gases in anymixture may vary widely, as one skilled in the art will recognize.Typically, however, at least about 0.01% of the total amount of the gasis a substantially insoluble gas, more preferably at least about 0.1%,even more preferably at least about 1%, and most preferably at leastabout 10%. Suitable ranges of substantially insoluble gas vary,depending upon various factors such as the soluble gas to beadditionally employed, the type of lipid, the particular application,etc. Exemplary ranges include between about 0.01% to about 99%substantially insoluble gas, preferably between about 1% and about 95%,more preferably between about 10% and about 90%, and most preferablybetween about 30% and about 85%.

For other uses beyond diagnostic ultrasound imaging, such as uses indiagnostic magnetic resonance imaging (MRI), paramagnetic gases such asthe strongly paramagnetic oxygen 17 gas (¹⁷ O₂), neon, xenon, helium,argon (especially rubidium enriched hyperpolarized xenon gas), or oxygen(which is still, albeit less strongly, paramagnetic), for example, arepreferably used to fill the headspace, although other gases may be alsoused. Most preferably, ¹⁷ O₂ gas, neon, rubidium enriched hyperpolarizedxenon gas, or oxygen gas is combined with a substantially insoluble gassuch as, for example, a perfluorocarbon gas. Paramagnetic gases are wellknown in the art and suitable paramagnetic gases will be readilyapparent to those skilled in the art. The most preferred gas for MRIapplications, whether used alone or in combination with another gas, is¹⁷ O₂.

By using a combination of gases, the ¹⁷ O₂ or other paramagnetic gasprovides the optimal contrast and the perfluorocarbon stabilizes the ¹⁷O₂ gas within the entrapped gas after shaking. Without the addition ofthe perfluorocarbon gas, gases such as ¹⁷ O₂ is generally much lesseffective, since because of its solubility it diffuses out of the lipidentrapment after intravenous injection. Additionally ¹⁷ O₂ gas is quiteexpensive. Combining the perfluorocarbon gas with ¹⁷ O₂ gas greatlyincreases the efficacy of the product and decreases the cost throughmore efficient use of the costly ¹⁷ O₂ gas. Similarly, other gases withdesirable paramagnetic properties, such as neon, may be mixed with theperfluorocarbon gases.

As Table 5, below, reveals, a wide variety of different gases may beused in MR imaging application. In Table 5, the R2 (1/T2/mmol/L.sec⁻¹)for different gases in gas-filled vesicles are shown. As Table 5 shows,there are dramatic differences in the relaxivity of the differentgas-filled vesicles, the higher the R2 relaxation values indicating themore effective the vesicles are as MR imaging agents. Of the gasesshown, air has the highest R2 value. It is believed that air is thehighest because of the paramagnetic effect of the oxygen in air. Pureoxygen, however, is somewhat less effective, likely due to the highersolubility of the oxygen and equilibration of oxygen into the aqueousmilieu surrounding the vesicles. With air, the nitrogen (air is about80% nitrogen) helps to stabilize the oxygen within the vesicles.Nitrogen has much less water solubility than air. As noted above, PFP orother perfluorocarbon gases may be mixed with a more magnetically activegas such as air, oxygen, ¹⁷ O₂ or rubidium enriched hyperpolarizedxenon. In so doing, stable highly magnetically active gas-filledvesicles may be prepared.

                  TABLE 5                                                         ______________________________________                                        Size Distribution and Relaxivity                                                         Number                                                                        Weighted                                                                      Distribution                                                                             Volume Weighted                                         Gas        (μm)    Distribution (μm)                                                                      R.sub.2                                     ______________________________________                                        Nitrogen   6.96 ± 0.63                                                                           31.08 ± 7.42                                                                           474.6 ± 59.9                             Sulfur Hexafluoride                                                                      4.31 ± 0.13                                                                           44.25 ± 1.23                                                                           319.3 ± 42.5                             Xenon(Rb)  7.02 ± 1.19                                                                           160.90 ± 92.46                                                                         191.2 ± 30.8                             Argon      S.14 ± 0.49                                                                            41.45 ± 13.02                                                                         55.29 ± 41.3                             Air        6.05 ± 1.05                                                                           23.28 ± 0.41                                                                           1510.4 ± 0.41                            Perfluoropropane                                                                         4.24 ± 0.72                                                                            49.88 ± 11.11                                                                         732.4 ± 31.8                             Oxygen     7.26 ± 0.98                                                                           30.99 ± 3.90                                                                           732.4 ± 73.9                             Neon       7.92 ± 0.71                                                                           26.20 ± 1.03                                                                           595.1 ± 97.2                             Perfluorobutane                                                                          5.88 ± 0.36                                                                           51.25 ± 3.97                                                                           550.1 ± 45.5                             ______________________________________                                    

The headspace of the container may be filled with the gas at ambient,decreased or increased pressure, as desired.

In the container of the invention, the gaseous phase is substantiallyseparate from the aqueous suspension phase. By substantially separate,it is meant that less than about 50% of the gas is combined with theaqueous suspension phase, prior to shaking. Preferably, less than about40%, more preferably less than about 30%, even more preferably less thanabout 20%, and most preferably less than about 10% of the gas iscombined with the aqueous suspension phase. The gaseous phase is keptsubstantially separate from the aqueous suspension phase, until aboutthe time of use, at which time the container is shaken and the gaseousphase and aqueous suspension phase combined to form an aqueoussuspension of gas-filled vesicles. In this fashion, an excellentcontrast agent for ultrasonic or magnetic resonance imaging is produced.Moreover, since the contrast agent is prepared immediately prior to use,shelf-life stability problems are minimized.

III. Container Volume and Headspace

It has been discovered that the size of the headspace of gas may also beused to affect gas-filled vesicle size. Since a larger headspacecontains proportionately more gas relative to the size of the aqueousphase, large headspaces will generally produce larger vesicles thansmaller sized headspaces. Therefore, the headspace, expressed as apercentage of the total volume of the vessel, should not exceed amaximum value. Moreover, too small a headspace will not allow sufficientroom for the fluid to move during the shaking to efficiently formvesicles.

For example, it is a discovery of this invention that when using vialsof 3.7 ml actual volume (Wheaton 300 Borosilicate glass, WheatonIndustries, Millville, N.J., referred to as 2 ml nominal size,diameter×height=15 mm×32 mm), the volume of the gas-containing headspaceis preferably between about 10% and about 60% of the total volume of thevial. Generally, the gas-containing headspace in a vial is between about10% and about 80% of the total volume of that vial, although dependingupon the particular circumstances and desired application, more or lessgas may be appropriate. More preferably, the headspace comprises betweenabout 30% and about 70% of the total volume. In general, it has beenfound that the most preferred volume of gas-containing headspace isabout 60% of the total volume of the container.

IV. Optimum Values for the Shaking Parameters

A. Shape of the Travel Path and Amplitude of Shaking

As previously discussed, in addition to the compositions of the aqueoussuspension and gaseous phases, the specific manner in which the vesselcontaining these phases is shaken will affect the vesicle sizedistribution. The optimal shaking conditions can be defined by referenceto four parameters--the shape of the path traveled by the containerduring the shaking, the amplitude of the shaking motion, the frequencyof the shaking, and the duration of the shaking.

It has been found that the path traveled by the container during theshaking is especially significant in the formation of proper sizedvesicles. In particular, it has been found that small vesicles can beproduced in a minimum amount of time when the shaking takes the form ofreciprocal motion. Other types of shaking, such as vortexing, can alsoproduce small vesicles. However, reciprocal shaking greatly reduces theduration of the shaking that is necessary to achieve a highconcentration of small vesicles.

The inventors have found that vesicles of small size are obtained in arelatively short period of time--i.e., 2 minutes or less--when theshaking amplitude--specifically, the length C of the reciprocal pathtraveled by the container during the shaking--is at least 0.3 cm. Ingeneral, the larger the amplitude of the shaking, the smaller thevesicles. However, as discussed below, the frequency of the shaking isalso an important parameter. Since practical considerations associatedwith the shaking equipment will typically result in a drop in shakingfrequency to undesirably low levels when the shaking amplitude isincreased beyond a certain maximum amount, the amplitude should bemaintained sufficiently low to ensure that the shaking frequency remainsadequate. For the Wig-L-Bug™ model 3110B shaking device, this maximumamplitude is approximately 2.5 cm.

The inventors have also found that it is preferable that the reciprocalmotion occur along an arcuate path 20, as shown in FIG. 4, wherein theamplitude of the shaking motion is denoted C, since high frequencyshaking motion is more readily accomplished in this manner. In thepreferred embodiment of the invention, the arcuate path 20 is defined bya radius of curvature L, which is formed by a shaker arm of length L.Preferably, the shaker arm 7 has a length L of at least 6 cm and rotatesthrough an angle θ of at least 3°. As discussed further below, accordingto the preferred embodiment of the invention, the angle of rotation ofthe shaker arm θ is achieved by employing a bearing having an offsetangle equal to θ. Further, the length L of the shaker arm is defined asthe distance from the center line of an eccentric bushing 40 on whichthe shaker arm 7 bearing 50 is mounted, as discussed further below, tothe centerline of the container 9, as shown in FIG. 3.

The use of longer shaker arm lengths L and larger angles of rotation θwill increase the amplitude of the shaking and, therefore, willgenerally reduce vesicle size. However, as discussed above, the maximumvalues for the shaker arm length L and the angle of rotation θ employedshould be limited to ensure that the amplitude of shaking C does notbecome so large that an inadequate shaking frequency results. Inaddition, mechanical considerations will also limit the size of theangle of rotation of the shaker arm 7. For the Wig-L-Bug™ model 3110B,the maximum shaker arm length and angle of rotation that should beemployed are approximately 15 cm and approximately 9°, respectively.

Additionally, it is preferred that the shaking device superimpose areciprocal motion in a second, approximately perpendicular, directiononto the reciprocal motion in the first direction. Preferably, theamplitude of shaking in the second direction C' is at leastapproximately one tenth that of the amplitude of shaking in the firstdirection C. For purposes of description, the first direction ofreciprocating motion will be referred to as the longitudinal directionand the second direction of reciprocating motion will be referred to asthe transverse direction.

Optimally, the timing of the motions in the longitudinal and transversedirections are adjusted so that the summation of the motions in the twodirections results in the container 9 shaking in a figure-8 pattern.

Based on the foregoing, the preferred shaking path described by thecontainer 9 when attached to the end of the shaker arm 7 of the shakingdevice 1 of the current invention is shown in FIGS. 4 and 5. As shown inFIG. 5, according to the current invention, the shaker arm 7 impartsmotion to the container 9 in the transverse direction as it move backand forth in the longitudinal direction in such a way that a point onthe container 9 travels in a figure-8 pattern 20. The length of thefigure-8 is the amplitude in the longitudinal direction C and the widthof the figure-8 is the amplitude of the shaking in the transversedirection C'. When viewed from the side, as shown in FIG. 4, the path isarcuate in the longitudinal direction--specifically, an arc having aradius of curvature that is equal to the length L of the shaker arm 7.The arc length C is the product of the shaker arm length L and the angleθ encompassed by the shaker arm rotation in the longitudinal plane,expressed in radians--that is, C=Lθ.

Preferably, the figure-8 pattern is comprised of approximately twostraight sections 21 that intersect at an angle θ and two approximatelyhalf-circle sections 22. As discussed further below, in the preferredembodiment of the invention, the angle φ formed by the figure-8 patternis approximately equal to the angle of rotation of the shaker arm 7 inthe longitudinal direction θ. As discussed below, this is accomplishedby applying sufficient force to the shaker arm 7 from a spring 46 so asto maintain the shaker arm essentially in a vertical orientation in thetransverse plane during the shaking, as shown in FIGS. 9 and 10. If thespring tension is adjusted to permit the shaker arm 7 to rotate throughan angle in the transverse plane ω, as shown in FIG. 12, then the angleφ of the figure-8 shaking pattern experienced by the container 9 will begreater than θ.

If φ equals θ, then the total distance D traveled in one circuit aroundthe path 20 will be a function of two variables--the length of theshaker arm L and the angle θ described by the shaker arm as it travelsin the longitudinal plane. This distance D can be approximated by theequation:

    D=2L[(2 sinθ/2+π tan.sup.2 9/2)/(1+tan 6/2)].

Since, preferably, the length L is at least 6 cm and the angle θ is atleast 3°, the distance D should preferably be at least about 0.6 cm.

Further, given that φ=θ, the amplitude of shaking in the transversedirection C' will be a function of the amplitude in the longitudinaldirection C and the angle θ, and can be approximated by the equation:

    C'=(2C tan θ/2)/(1 +tan θ/2)

Since, preferably, the amplitude of the shaking in the longitudinaldirection C is at least about 0.3 cm and the angle ∂ is at least about3°, the amplitude in the transverse direction C' should preferably be atleast about 0.02 cm.

The optimum values for the amplitude and shape of the shaking motiondiscussed above were arrived at based on a series of tests, discussedbelow in sub-section C.

B. Frequency and Duration of Shaking

In addition to the shape and amplitude of the shaking motion, thefrequency of the shaking is also an important parameter in formingproper sized vesicles. The shaking frequency is quantified in terms ofthe revolutions per minute ("RPM") experienced by the shaker arm 7 andis defined as the number of times the shaker arm, and, therefore thecontainer 9 attached to it, traverses the entirety of the shaking pathin one minute. Thus, in the preferred embodiment of the invention,shaking at a frequency of 3600 RPM means that the container 9 undergoesshaking motion around the figure-8 path 20 thirty six hundred times inone minute, or sixty times in one second.

It has been found that vesicles can be made using shaking frequencies inthe range of 100 RPM to 10,000 RPM. However, it has been found thatthere is a minimum shaking frequency that will result in the productionof optimally sized vesicles within a relatively short period of time. Asdiscussed in section C, below, in has been found that this minimumfrequency is approximately 2800 RPM. Although, in general, increasingthe shaking frequency will reduce vesicle size, the limitations of theshaking device will typically set the maximum obtainable frequency. Forthe Wig-L-Bug™, the maximum obtainable frequency is about 3300 RPM.

At frequencies in the range of 2800 to 3300 RPM, the optimum duration ofthe shaking is at least approximately 60 seconds. However, the optimalduration of the shaking is related to the frequency and may be lower athigher frequencies. Thus, for example, at 4500 RPM the optimal durationof shaking is only 50 seconds.

C. Test Results

The optimum value for the shaking frequency, as well as the shape andamplitude of the shaking motion, were developed through a series oftests, as discussed below.

A first series of tests were conducted to determine the effect ofshaking frequency on vesicle size. One mg mL⁻¹ samples of lipidconsisting of dipalmitoylphosphatidylcholine (DPPC) (Avanti PolarLipids, Alabaster, Ala.), dipalmitoylphosphatidic acid (DOPPA) (AvantiPolar Lipids, Alabaster, Ala.), and dipalmitoylphosphatidylethanolaminecovalently bound to polyethyleneglycol monomethyl ether of molecularweight 5000, (DPPE PEG-5000) (Avanti Polar Lipids, Alabaster, Ala.), ina mole ratio of 82 mole %:10 mole %:8 mole % respectively, were added toa diluent consisting of normal saline, glycerol (Spectrum Chemical Co.,Gardena, Calif.), and propylene glycol (Spectrum Chemical Co., Garden,Calif.), (8:1:1, v:v:v). The samples were then heated to 45° C. for 10minutes then allowed to equilibrate to room temperature (25° C.).

The samples were then added to nominal 2.0 mL borosilicate vials (VWRScientific, Boston, Mass.) of the type shown in FIG. 1 (actual volume3.7 mL). The vials were then sealed with a butyl rubber stopper andclosed to a gas-tight fit with an aluminum crimp. The headspace in thevials was approximately 60% of the total volume of the vials. Sampleswere then purged with perfluoropropane (Flura Corporation, Nashville,Tenn.) and placed on the shaking device shown in FIG. 3, which isdiscussed further in section V.

The containers were shaken for 2 minutes using the figure-8 type motionshown in FIGS. 4 and 5. The length of the shaker arm L was 7.7 cm andthe bearing offset angle d and, therefore, the angle of rotation of theshaker arm in the longitudinal plane, was 6°. Using the relationshipsdiscussed above, it was determined that the amplitude of shaking in thelongitudinal and transverse directions C and C' were approximately 0.8cm and 0.1 cm, respectively.

Shaking frequencies of 1500, 2800 and 3300 RPM were used, measured via aCode-Palmer Model 08210 Pistol Grip tachometer (Code-Palmer, Nile,Ill.). Sizing was determined by small particle optical sizing on aParticle Sizing System light obscuration particle sizer (Santa Barbara,Calif.).

Table 6 shows the results of these tests and demonstrates the effectthat shaking frequency has on the resultant average vesicle size.

                  TABLE 6                                                         ______________________________________                                        Effect of Shaking Frequency on Average Vesicle Size                           Frequency (RPM)                                                                             Average Vesicle Size                                            ______________________________________                                        1500          3.4 μm                                                       2800          3.3 μm                                                       3300          2.9 μm                                                       ______________________________________                                    

As can be seen, shaking at a frequency in excess of 2800 RPM greatlyreduces the average vesicle size obtained after 2 minutes of shaking.

A second set of tests were conducted to determine the effect on vesiclesize of increasing the shaker arm length L, and, therefore, the shakingamplitude in the longitudinal and transverse directions C and C', aswell as the shaking distance per cycle D. The tests were conducted thesame as those discussed above except that the containers were shaken for60 seconds using shaker arm lengths L over the range of 6.7 to 14.8 cm.

The variations in the shaker arm length resulted in variations inshaking frequency over the range of 2250 to 3260 RPM, with the shakingfrequency decreasing as the shaker arm length increased. The variationin shaking frequency with the shaker arm length L and the shaker armrotation angle θ is shown in FIG. 13. Thus, for example, when a shakerarm length L of 6.7 cm and an angle of rotation θ of 6° were used, thefrequency of shaking was approximately 3200 RPM, whereas when the shakerarm length was increased to 13.8 cm, while maintaining the same angle ofrotation, the frequency dropped to about 2700 RPM.

The results of this series of tests are shown in FIGS. 14(a)-(c). Asshown in FIG. 14(a), with a angle of rotation in the longitudinal planeθ of 6°, at least 98% of the vesicles are below 10 microns whenever theshaker arm length L is 7.7 cm or greater--that is, when the amplitudesof shaking in the longitudinal direction C is greater than 0.8 cm.Moreover, the percentage of vesicles below 10 μm reaches a plateau ofabout 99 to 99.5% at shaker arm lengths L of 9.8 cm and above--that is,when the amplitudes of shaking in the longitudinal direction is 1.0 cmand above. The number weighted mean size of the vesicles reaches aplateau of about 2 μm at these same conditions, as shown in FIG. 14(b).

Although the general effect of increasing shaking frequency is to reducevesicle size when all other variables are held constant, as previouslydiscussed and shown in Table 6, these data show that increasing theshaking amplitude by increasing the shaker arm length reduces the sizeof the vesicles even when such increases are combined with reductions inshaking frequency, as shown in FIG. 13.

As shown in FIG. 14(c), more than 400×10⁶ vesicle per mL were obtainedat all shaker arm lengths and, in fact, the use of shaker arm lengths inthe range of about to 12 cm resulted in the production of more than1000×10⁶ vesicle per mL. However, as the shaker arm length is increasedabove about 12 cm, the particles per mL began dropping and reached800×10⁶ vesicles per mL at 14.8 cm. Although not shown in FIG. 13, witha 14.8 cm shaker arm length and a 6° shaker arm angle of rotation, thefrequency was determined to be only 2550 RPM. Thus, the drop in theconcentration of vesicles produced with a 14.8 cm arm length is thoughtto be due to the drop in shaking frequency that accompanies increases inshaking amplitude, as previously discussed. Therefore, these dataindicate that when using a Wig-L-Bug™ shaking device, the shaker armlength should preferably be less than approximately 15 cm to maximizevesicle concentration.

A third series of tests were performed using the same materials andprocedure discussed above except that the bearing offset angle and,therefore, the angle of the shaker arm rotation in the longitudinaldirection θ, was increased from 6° to 9°, thereby increasing theamplitude of shaking in the longitudinal direction. In addition, shakerarm lengths in excess of 11.8 cm were not used. The results of thesetests are shown in FIGS. 15(a)-(c), along with the results of thepreviously discussed set of tests for comparison.

As can be seen in FIG. 15(a), increasing the angle of rotation of theshaker arm θ from 6° to 9° reduces vesicle size, even though it also hasthe effect of reducing the shaking frequency, as shown in FIG. 13. Thus,with a 9° shaker arm angle of rotation, even a shaker arm length of only6.7 cm results in over 99.5% of the vesicles being below 10 μm and amean size of about 2 μm. In addition, in excess of 1000×10⁶ vesicle permL were obtained at all shaker arm lengths, as shown in FIG. 15(c).

Another series of tests were performed using the same materials andprocedure discussed above except that bearing offset angles and,therefore, angles of the shaker arm rotation in the longitudinaldirection θ, of 3°, 5.2°, 6°, 7.8°, and 9° were used along with shakerarm lengths L between 6.7 cm and 13.8 cm (increasing in approximately 1cm increments). The total length D of the shaking path 20 was estimatedat each point. The frequency as a function of total path length is shownin FIG. 17. The results are shown in FIGS. 16(a)-(c) as a function ofthe total path length D. As can be seen, under all of the conditionstested--i.e., at total path lengths D of 0.7 cm and above--more than 95%of the vesicles were less than 10 μm and the concentration of vesiclesproduced was more than 100×10⁶ per mL. Further, under all conditions inwhich the total path length D was 2.19 cm or greater, more than 98% ofthe vesicles were less than 10 μm. This suggests that the total pathlength of the shaking motion should be at least 0.7 cm and, morepreferably, at least 2.2 cm.

Thus, the foregoing shows that vesicles of small size can be obtained inabout two minutes or less when reciprocal shaking is conducted such thatthe frequency of shaking is at least approximately 2800 RPM. Inaddition, the shaking motion should be accomplished in two substantiallyperpendicular directions, and, more preferably, in a figure-8 pattern.Further, the amplitude of shaking in the major direction should be atleast 0.3 cm and, more preferably at least 0.8 cm, or the total lengthof the shaking path should be at least 0.7 cm and, more preferably, atleast 2.2 cm.

V. The Apparatus of the Invention

A. The Preferred Shaking Device

The preferred shaking device 1 of the current invention is shown inFIGS. 2 and 3. The apparatus is comprised of a base 2 and a hingedsafety cover 3. A start-stop button 6 and a speed control dial 5 aremounted on a housing 4 that encloses the base 2. An arm 7 projectsupward through an opening 12 in the upper portion of the housing 4.Turning dial 5 clockwise increases the shaking speed while turning thedial counter-clockwise decreases the shaking speed.

According to the current invention, a mounting bracket 8 is attached tothe distal end of the arm 7 that allows the container 9, discussedfurther below, to be secured to the arm. The bracket 8 is fitted withseveral spring clips 11 and 12 that hold the container 9 securely inplace. Alternatively, a thumb screw type bracket could also be used toprovide even more secure attachment of the container 9. As shown in FIG.3, the bracket may be oriented at an angle δ to the horizontal so that,when installed in the device 1, the axis of the container 9 will also beoriented at an angle δ to the horizontal. Preferably, the angle δ is inthe range -5° to +5°, and most preferably is about 0°. In use, thecontainer 9 is secured to the bracket 8 and the shaker device 1 isoperated to vigorously shake the container along the path of travelshown in FIGS. 4 and 5.

FIGS. 6-12 show the major internal components of the shaker device 1according to the current invention. As can be seen, the shaker arm 7 isrotatably mounted onto the shaft 42 of an electric motor 44. As shownbest in FIGS. 6 and 9, a cylindrical sleeve 41 is formed at the proximalend of the shaker arm 7. The sleeve 41 houses a bearing 50 that supportsa cylindrical eccentric bushing 40. The bushing 40, shown best in FIG.11, is fixedly attached to the shaft 42 --for example, by being pressedonto or integrally formed with the shaft--and rotates within the bearing50.

As shown best in FIG. 11, one end 43 of the bushing 40 is eccentric withrespect to the shaft 42 while the other end 45 of the bushing isconcentric with the shaft. Consequently, as shown best in FIG. 7, thecenter line of the bushing 40 forms an acute angle θ/2 with the centerline of shaft 42. The angle θ is referred to as the bearing offsetangle. As previously discussed, the bearing offset angle θ is preferablyat least about 3°. As shown in FIGS. 7 and 8, as the shaft 42 andbushing 40 rotate 360°, the shaker arm 7 rotates back and forth in thelongitudinal plane by an angle that is equal to θ (when the bushing 40is in the orientation shown in FIG. 8, the position of the shaker arm 7is as shown in phantom in FIG. 7). Thus, rotary motion of the shaft 42rotates the sleeve 41 in the longitudinal plane and imparts rectilinearmotion along an arcuate path to the distal end of the shaker arm 7 towhich the container 9 is secured, as shown in FIG. 4.

Due to the eccentric nature of the bushing 40, rotation of the shaft 42also tends to rotate the sleeve 41 of the shaker arm 7 through thebearing offset angle θ in the transverse direction as well, as shown inFIG. 10 (the position of the shaker arm when the bushing has beenrotated 180° is shown in phantom in FIG. 10). Thus, if the rotation ofthe shaft 42 were clockwise when viewed from left to right in FIG. 7,then the orientation of the shaker arm 7 when the eccentric bushing 40is at 0° is shown by the solid lines in FIG. 7, the orientation when thebushing is at 90° is shown in phantom in FIG. 10, the orientation whenthe bushing is at 180° is shown in FIG. 8, and the orientation when thebushing is at 270° is shown by the solid lines in FIG. 10. Thus, as theeccentric bushing 40 rotates 360° within the bearing 50, the shaker arm7 imparts a shaking motion to the container 9 in both the longitudinaland transverse directions so as to achieve the figure-8 patternpreviously discussed.

A spring 46 extends from bottom dead center of the sleeve 41 to the baseplate 5 of the shaker housing, as shown in FIG. 6. Tension on the spring46 acts to keep the shaker arm 7 in the upright position as the bushing40 rotates. Preferably, the spring 46 has sufficient tension so that theshaker arm 7 remains essentially vertically oriented in the transverseplane, as shown in FIGS. 9(a) and (b), although it departs from thevertical by θ/2 in the longitudinal plane, as shown in FIG. 7. Utilizinga spring 46 having a lesser spring constant will allow the shaker arm 7to rotate in the transverse plane through an angle ω, as shown in FIG.12. This has the effect of increasing the amplitude in the transversedirection C'.

Preferably, the shaking device 1 is constructed by modifying acommercially available shaking device manufactured by Crescent DentalManufacturing, Inc., 7750 West 47th Street, Lyons, Ill. 60534 under thename Wig-L-Bug™ 3110B shaker. Such Wig-L-Bug™ devices employ a figure-8type of shaking pattern and are sold having a shaker arm with a length Lof 4 cm, a bearing offset angle and, therefore, a shaker arm angle ofrotation in the longitudinal direction θ of 6°, and operate at a fixedspeed of 3200 RPM. Further, the shaker arm on the Wig-L-Bug features apair of spoons to hold the samples.

Thus, the shaking apparatus of the current invention may be created bymodifying a Wig-L-Bug™ 3110B shaker to incorporate the container 9, intowhich the aqueous suspension and gaseous phases have been added, aspreviously discussed, onto the distal end of the arm 7. Preferably, theWig-L-Bug™ shaker is also modified so as to incorporate the mountingbracket 8 for securing the container 9 onto the shaker arm 7, as shownin FIGS. 3 and 6. In addition, depending on the composition of theaqueous and gases phases, the size of the container, etc., optimalresults may be obtained by further modifying the Wig-L-Bug™ so as to (i)provide shaking at a frequency other than 3200 RPM or to allow operationover a range of shaking frequencies, (ii) employ a shaker arm lengthother than 4 cm, or (iii) employ a bearing offset angle θ other than 6°by modifying the offset bushing 40.

Other types of reciprocal shaking devices can also be used in thepractice of the current invention, most preferably, devices which imparta figure-8 shaking motion. In addition to the Wig-L-Bug™, such devicesinclude (i) the Mixomat, sold by Degussa AG, Frankfurt, Germany, (ii)the Capmix, sold by Espe Fabrik Pharmazeutischer Praeparate GMBH & Co.,Seefeld, Oberay Germany, (iii) the Silamat Plus, sold by Vivadent,Lechtenstein, and (iv) the Vibros, sold by Quayle Dental, Sussex,England.

FIGS. 18(a)-(c) show the results of tests on the Mixomat and Capmixcompared to test results obtained from a Wig-L-Bug™ 3110B using the samematerials and procedures previously discussed with respect to the testresults shown in FIGS. 13-17, and a shaking duration of 60 seconds, withthe Wig-L-Big™ operating at a frequency of 3200 RPM, the Mixomatoperating at a frequency of 4100 RPM, and the Capmix operating at afrequency of 4500 RPM. As can be seen, in each instance, more than 98%of the vesicles were less than 10 μm and more than 800×10⁶ vesicles permL were produced.

B. The Preferred Container

According to the current invention, the container that is secured to theshaking device 1 may take a variety of different forms. A preferredcontainer 9 is shown in FIG. 1 and comprises a body 30 and a gas tightcap 10. When filled, the container 9 forms a headspace of gas 32 and anaqueous suspension phase 34 substantially separate from one another.Alternatively, the container may take the form of a pre-filled syringe,which may, if desired, be fitted with one or more filters. Accordingly,the term container, as used herein, includes a syringe. Syringes, filledwith an aqueous phase and a headspace of a pre-selected gas, arepreferably mounted on the shaking device 1 with their long axes orientedin the transverse direction--that is, perpendicular to the arc length C.After shaking, the gas-filled vesicles are produced in the syringe,ready to use. Regardless of the type of container used, it is preferablysterile, along with its contents.

Although, in general, the invention is practiced with sterile containerswherein the aqueous phase is already present within the container, forselected applications, the stabilizing media may be stored within thecontainer in a dried or lyophilized state. In this case the aqueoussolution, e.g. sterile phosphate buffered saline, is added to thesterile container immediately prior to shaking. In so doing, therehydrated stabilizing media within the aqueous phase will againinteract with the gas headspace during shaking so as to producegas-filled vesicles as above. Rehydration of a dried or lyophilizedsuspending medium necessarily further complicates the product and isgenerally undesired but for certain preparations may be useful forfurther extending the shelf life of the product. For example, certaintherapeutic agents such as cyclophosphamide, peptides, and geneticmaterials (such as DNA), might be hydrolyzed on long term aqueousstorage. Rehydration of a previously lyophilized sample to form theaqueous phase and headspace prior to shaking can make it practical toproduce gas-filled vesicles containing compounds which otherwise mightnot have sufficient shelf life.

A variety of different materials may be used to produce the container,such as glass, borosilicate glass, silicate glass, polyethylene,polypropylene, polytetrafluoroethylene, polyacrylates, polystyrene, orother plastics. The preferred containers are either gas impermeable orwrapped within an outer gas impermeable barrier prior to filling withgas. This is, or course, desirable to maintain the integrity of thepre-selected gas within the vessel. Examples of syringe materials havinggas-tight capabilities may include but are by no means limited to glasssilicates or borosilicates, fitted with silica-fused syringes orluer-lock type syringes, and teflon-tipped or teflon-coated plungers.

The size of the container, more specifically, its weight, will affectthe size of the gas-filled vesicles. Shaking devices will generallyshake more slowly as the weight of the container increases beyond acertain level--for example, a Wig-L-Bug™ 3110B shakes more quickly witha 2 ml vial (actual volume 3.7 ml) than a 10 ml vial. Therefore, thevolume of the container should not exceed a certain amount depending onthe particular shaking device utilized.

Tests were performed on a Wig-L-Bug utilizing both a 10 ml clear vial(Wheaton Industries, Millville, N.J.) and a 2 ml (actual volume 3.7 ml)amber vial (Wheaton Industries, Millville, N.J.). Once again, the rateof shaking was measured, using a Code-Palmer Pistol Grip tachometer(Code-Palmer, Nile, Ill.). Table 7 lists the results, which demonstratethat increasing the capacity of the vial will decrease the shakingfrequency.

                  TABLE 7                                                         ______________________________________                                        Effect of Vial Size on Wig-L-Bug ™ Shaking Frequency                       Vial Size  Measured Frequency (RPM)                                           ______________________________________                                         2 ml vial 3250                                                               10 ml vial 2950                                                               ______________________________________                                    

As can be seen, a 2 mL nominal provides permits the use of a highshaking frequency on the Wig-L-Bug. With reference to the dimensionsshown in FIG. 1, a 2 mL nominal, 3.7 mL actual, container 9 preferablyhas a diameter D of approximately 0.7 inch, and overall height H_(O) ofapproximately 1.4 inch and a body height H_(B) of approximately 1 inch.

VI. Applications of the Vesicles Produced According to the CurrentInvention

The foregoing sets forth various parameters in determining gas-filledvesicle size. Vesicle size is of importance in terms of maximizingproduct efficacy and minimizing toxicity. Additionally the vesiclesshould be as flexible as possible both to maximize efficacy and tominimize adverse tissue interactions such as lodging in the lungs. Thepresent invention creates vesicles of the desired size with very thincompliant membranes. Because the vesicle membranes are so thin andcompliant, e.g. only 1 mg ml⁻¹ of lipid is necessary for stabilizing themembranes, it has been found that gas-filled vesicles of larger diametermay be used without producing pulmonary hypertension. For example, pigshave been administered doses up five time the necessary diagnosticimaging dose without any evidence of pulmonary hypertension. Bycomparison much lower doses of smaller diameter albumin coated airbubbles in these animals cause severe pulmonary hypertension. Becausethe vesicles of the present invention are so flexible and deformable,they easily slide through the lung capillaries. Additionally the coatingtechnologies employed with the present lipids (e.g. polyethyleneglycolbearing lipids) decreases adverse pulmonary interactions while at thesame time enhancing the in vitro and in vivo stability and efficacy ofthe product.

The size of gas-filled vesicles for use as general ultrasound contrastmedia should be as large as possible (without causing embolic effects)because backscatter or the ultrasound effect is proportional to theradius to the sixth power when frequencies are such that the gas-filledvesicles are in the Rayleigh scattering regime. For MRI, larger vesiclesof the invention are also preferred. The ability of the presentinvention to prepare and employ larger vesicle size with less potentialof toxic effects increases its efficacy relative to other products.

An additional parameter influencing ultrasound contrast is theelasticity of the vesicle membrane. The greater the elasticity thegreater the contrast effect. Because the present vesicles are coated byultra-thin membranes of lipid elasticity is quite similar to naked gasand reflectivity and contrast effect are maximized.

The shaking procedure of the present invention readily produces vesiclesfrom an aqueous phase and a headspace of gas within a sterile container.The invention is sufficient for producing vesicles with highly desirableproperties for ultrasonic or magnetic resonance imaging applications.For selected applications however, a filter may be employed to producevesicles with even more homogeneous size distributions and of desireddiameters.

For example for measuring in vivo pressures on ultrasound usinggas-filled vesicle harmonic phenomena, it may be useful to have verytightly defined vesicle diameters within a narrow range of sizes. Thisis readily accomplished by injecting the vesicles (produced by shakingthe container with aqueous phase and headspace of gas) through a filterof defined size. The resulting vesicles will be no larger than a veryclose approximation of the size of the filter pores in the filtermembrane. As noted above, for many ultrasonic or MRI applications, it isdesirable to have the gas-filled vesicles be as large as possible. Forcertain applications however, much smaller gas-filled vesicles may bedesirable. In targeting, for example, to tumors or other diseasedtissues, it may be necessary for the gas-filled vesicles to leave thevascular space and to enter the tissue interstitium. Much smallergas-filled vesicles may be useful for these applications. These smallergas-filled vesicles (e.g., appreciably under a micron in diameter) canto a large extent be produced by modifications in the compounds in theaqueous phase (composition and concentration), as well as the headspace(composition of gas and volume of headspace), but also by injectionthrough a filter. Very small gas-filled vesicles of substantiallyhomogeneous size may be produced by injecting through for example a 0.22micron filter. The resulting nanometer sized gas-filled vesicles maythen have desirable properties for targeting.

The above examples of lipid suspensions may also be sterilized viaautoclave without appreciable change in the size of the suspensions.Sterilization of the contrast medium may be accomplished by autoclaveand/or sterile filtration performed either before or after the shakingstep, or by other means known to those skilled in the art.

After filling the containers with the aqueous phase and the headspace ofthe pre-selected gas the sealed bottles may be stored indefinitely.There need be no particles to precipitate, gas-filled vesicles to burstor other untoward interactions between gas-filled vesicles, particles,colloids or emulsions. The shelf life of the container filled with theaqueous phase and headspace of gas depends only on the stability of thecompounds within the aqueous phase. These properties of long shelf lifeand sterilizability confer substantial advantages to the presentinvention over the prior art. The problem of stability, such as withaggregation and precipitation of particles, which was so common in thefield of ultrasound contrast media have been addressed herein.

The gas-filled vesicles which are produced by shaking of the multi-phasecontainer of the invention have been found to have excellent utility ascontrast agents for diagnostic imaging, such as ultrasound or magneticresonance imaging. The vesicles are useful in imaging a patientgenerally, and/or in specifically diagnosing the presence of diseasedtissue in a patient. The imaging process may be carried out byadministering a gas-filled vesicle of the invention to a patient, andthen scanning the patient using ultrasound or magnetic resonance imagingto obtain visible images of an internal region of a patient and/or ofany diseased tissue in that region. By region of a patient, it is meantthe whole patient, or a particular area or portion of the patient. Theliposomal contrast agent may be employed to provide images of thevasculature, heart, liver, and spleen, and in imaging thegastrointestinal region or other body cavities, or in other ways as willbe readily apparent to those skilled in the art, such as in tissuecharacterization, blood pool imaging, etc. Any of the various types ofultrasound or magnetic resonance imaging devices can be employed in thepractice of the invention, the particular type or model of the devicenot being critical to the method of the invention.

The gas-filled vesicles of the invention may also be employed to delivera wide variety of therapeutics to a patient for the treatment of variousdiseases, maladies or afflictions, as one skilled in the art willrecognize.

Also, magnetically active vesicles may be used for estimating pressureby MRI. The vesicles increase the bulk susceptibility and, accordingly,increase T₂ relaxation but even more so for T₂ * relaxation. Because theeffects of static field gradients are mainly compensated in spin echoexperiments (by virtue of the 180° radiofrequency refocusing pulse) theeffect of the vesicles is less marked on T₂ than T₂ * weighted pulsesequences where static field effects are not compensated. Increasingpressure results in loss of vesicles or vesicle disruption (for moresoluble gases) as well as a decrease in vesicle diameter. Accordingly,1/T₂ decreases with increasing pressure. After release of pressure someof the remaining vesicles re-expand and 1/T₂ increases again slightly.Vesicles composed of about 80% PFP with 20% air show enhanced stabilityand a slight fall in 1/T₂ with pressure which returns to baseline afterrelease of pressure (i.e., the vesicles are stable but show a slight1/T₂ pressure effect). When gradient echo images are obtained and signalintensity measured these effects are much more marked. Signal intensityincreases with increasing pressure (1/T₂ * decreases with increasedpressure). Because the experiment is performed relatively quickly (ittakes less than a tenth the time to perform the gradient echo imagesthan to measure T₂). The duration of exposure to pressure is much lessand the nitrogen filled vesicles return nearly to baseline afterpressure release (i.e. there is very little loss of vesicles).Accordingly, the signal intensity on gradient echo falls back nearly tobaseline at return to ambient pressure. For measurement of pressure byMRI, the vesicles may be designed either to fall apart with increasingpressure or to be stable but decrease vesicle diameter with increasingpressure.

Because on MRI vesicle radius affects 1/T₂ *, this relationship can beused to estimate pressure by MRI.

As one skilled in the art would recognize, administration of thegas-filled vesicles to the patient may be carried out in variousfashions, such as intravenously or intraarterially by injection, orally,or rectally. The useful dosage to be administered and the particularmode of administration will vary depending upon the age, weight and theparticular mammal and region thereof to be scanned or treated, and theparticular contrast medium or therapeutic to be employed. Typically,dosage is initiated at lower levels and increased until the desiredcontrast enhancement or therapeutic effect is achieved. The patient canbe any type of mammal, but most preferably is a human.

The disclosures of each of the patents and publications cited orreferred to herein are hereby incorporated herein by reference in theirentirety.

Various modifications of the invention in addition to those shown anddescribed herein will be apparent to those skilled in the art from theforegoing description. Such modifications are also intended to fallwithin the scope of the appended claims.

What is claimed:
 1. An apparatus for making vesicles, comprising:a) acontainer containing an aqueous suspension phase and a gaseous phasesubstantially separate from said aqueous suspension phase; b) a devicefor shaking said container by imparting a reciprocating motion theretoso as to form vesicles, said shaking device comprising (i) an arm havinga distal end to which said container is coupled, and (ii) means forpivoting said arm back and forth in first and second mutuallyperpendicular directions so as to reciprocate said container along anarcuate path, wherein said means for pivoting said arm comprises meansfor pivoting said arm at a frequency greater than approximately 2800 RPMand no greater than approximately 10,000 RPM.
 2. The apparatus accordingto claim 1, wherein said means for pivoting said arm comprises means forreciprocating said distal end of said arm at an amplitude of at leastapproximately 0.8 cm in said first direction.
 3. The apparatus accordingto claim 2, wherein said means for pivoting said arm further comprisesmeans for reciprocating said distal end of said arm at an amplitude nogreater than approximately 2.5 cm in said first direction.
 4. Theapparatus according to claim 1, wherein said means for pivoting said armcomprises means for reciprocating said distal end of said arm along apath having a total length of at least approximately 0.7 cm.
 5. Theapparatus according to claim 1, wherein the angle encompassed by saidarcuate path is at least approximately 3°.
 6. The apparatus according toclaim 5, wherein the angle encompassed by said arcuate path is nogreater than approximately 9°.
 7. The apparatus according to claim 1,wherein the radius of curvature of said arcuate path is at leastapproximately 6 cm.
 8. The apparatus according to claim 7, wherein theradius of curvature of said arcuate path is no greater thanapproximately 15 cm.
 9. The apparatus according to claim 1, wherein saidmeans for pivoting said arm further comprises means for reciprocatingsaid distal end of said arm along a path having approximately a figure-8pattern.
 10. The apparatus according to claim 9, wherein the totallength of said figure-8 pattern is at least approximately 0.7 cm. 11.The apparatus according to claim 1, wherein said aqueous suspensionphase comprises lipids.
 12. The apparatus according to claim 11, whereinsaid aqueous suspension phase comprises dipalmitoylphosphatidylcholine,dipalmitoylphosphatidic acid, and dipalmitoylphosphatidylethanolamine.13. The apparatus according to claim 12, wherein said gaseous phasecomprises a perfluorocarbon gas.
 14. The apparatus according to claim 1,wherein said gas initially occupies at least 10% of the volume of saidcontainer.
 15. An apparatus for making vesicles, comprising:a) acontainer containing an aqueous suspension phase and a gaseous phasesubstantially separate from said aqueous suspension phase; b) a shakingdevice for shaking said container so as to form vesicles therein, saidshaking device having (i) an arm having a distal end and a length of atleast approximately 6 cm, said arm being mounted so as to pivot in afirst direction through a first angle of at least approximately 6°, (ii)means for pivoting said arm back and forth in said first directionthrough said first angle so that said distal end of said armreciprocates at an amplitude of at least 0.8 cm along an arcuate path,and (iii) means for coupling said container to said arm.
 16. Theapparatus according to claim 1 wherein said vesicles comprise amonolayer.
 17. The apparatus according to claim 16 wherein saidmonolayer comprises a phospholipid.
 18. The apparatus according to claim17 wherein said gaseous phase is selected from the group consisting ofperfluoropropane, perfluorobutane, perfluoropentane, perfluorohexane,and sulfur hexafluoride.
 19. The apparatus according to claim 17 whereinsaid gaseous phase is perfluoropentane.
 20. The apparatus according toclaim 17 wherein gaseous phase is sulfur hexafluoride.
 21. The apparatusaccording to claim 17 wherein said gaseous phase is perfluoropropane.22. The apparatus according to claim 15 wherein said vesicles comprise amonolayer.
 23. The apparatus according to claim 22 wherein saidmonolayer comprises a phospholipid.
 24. The apparatus according to claim22 wherein said gaseous phase is selected from the group consisting ofperfluoropropane, perfluorobutane, perfluoropentane, perfluorohexane,and sulfur hexafluoride.
 25. The apparatus according to claim 22 whereinsaid monolayer comprises a phospholipid and said gaseous phase isperfluoropentane.
 26. The apparatus according to claim 22 wherein saidmonolayer comprises a phospholipid and said gaseous phase is sulfurhexafluoride.
 27. The apparatus according to claim 22 wherein saidmonolayer comprises a phospholipid and said gaseous phase isperfluoropropane.
 28. The apparatus according to claim 1 wherein saidvesicles comprise a polymer.
 29. The apparatus according to claim 28wherein said polymer comprises an acrylate.
 30. The apparatus accordingto claim 29 wherein said gas is air.
 31. The apparatus according toclaim 28 wherein said polymer comprises a methacrylate.
 32. Theapparatus according to claim 31 wherein said gas is air.
 33. Theapparatus according to claim 15 wherein said vesicles comprise apolymer.
 34. The apparatus according to claim 33 wherein said polymercomprises an acrylate.
 35. The apparatus according to claim 34 whereinsaid gas is air.
 36. The apparatus according to claim 33 wherein saidpolymer comprises a methacrylate.
 37. The apparatus according to claim36 wherein said gas is air.
 38. The apparatus according to claim 1wherein said vesicles comprise a polysaccharide.
 39. The apparatusaccording to claim 38 wherein said polysaccharide comprises galactose.40. The apparatus according to claim 39 wherein said gaseous phase isnitrogen.
 41. The apparatus according to claim 15 wherein said vesiclescomprise a polysaccharide.
 42. The apparatus according to claim 41wherein said polysaccharide comprises galactose.
 43. The apparatusaccording to claim 42 wherein said gaseous phase is nitrogen.
 44. Theapparatus according to claim 11 wherein said aqueous suspension phasecomprises liposomes.
 45. The apparatus according to claim 44 whereinsaid liposomes comprise cross-linked liposomes or polymerized liposomes.46. The apparatus according to claim 11 wherein said aqueous suspensionphase further comprises polyethylene glycol.
 47. The apparatus accordingto claim 2, wherein said means for pivoting said arm comprises means forreciprocating said distal end of said arm at an amplitude of at leastapproximately 0.02 cm in said second direction.
 48. The apparatusaccording to claim 15, wherein said arm is mounted so as to pivot in asecond direction through a second angle, said second direction beingperpendicular to said first direction, and wherein said pivoting meanshas means for pivoting said arm back and forth in said second directionthrough said second angle.